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. Author manuscript; available in PMC: 2016 Dec 23.
Published in final edited form as: Reproduction. 2011 Mar 9;141(6):849–856. doi: 10.1530/REP-10-0462

Functions of ectopically transplanted invasive horse trophoblast

Amanda M de Mestre 1,2, David Hanlon 3, A Paige Adams 1, Erin Runcan 1, Jane C Leadbeater 1, Hollis N Erb 4, Christina C Costa 1, Donald Miller 1, W R Allen 5, Douglas F Antczak 1,6
PMCID: PMC5181105  NIHMSID: NIHMS518968  PMID: 21389079

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 Gonadotrophin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and (iii) if 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% CI 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 three 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.

Keywords: trophoblast, transplantation, immunology, equine chorionic gonadotrophin

Introduction

In the pregnant state in mammals a variety of mechanisms operate to ensure that the semi-allogeneic fetus is not rejected by the maternal immune system (Moffett & Loke 2006, Seavey & Mosmann 2008, Noronha & Antczak 2010). Indeed, the use of embryo transfer has demonstrated that fully allogeneic and even xenogeneic conceptuses (Allen & Short 1997) can survive in a foreign uterine environment. Importantly, there is abundant evidence for simultaneous maternal immune recognition (awareness) and immune regulation that permits peaceful co-existence between mothers and their fetuses for the duration of gestation (Tafuri et al. 1995, Robertson & Sharkey 2001, Aluvihare et al. 2004). Critical to this interaction are the trophoblast cells that comprise the outer layer of the mammalian placenta. Trophoblast cells can modulate immunity both directly and indirectly, for example via the expression of molecules such as FasL, and the secretion of immunosuppressive molecules (Flaminio & Antczak 2005, Fraccaroli et al. 2009). Trophoblast cells also have the capacity to produce high concentrations of molecules that are secreted into the maternal circulation and act on maternal tissues in normal pregnancy (Huppertz 2008).

Transplantation of trophoblast tissues offers an approach to distinguish between immunoprotective mechanisms that are innate to the trophoblast from those related to the endocrine milieu of pregnancy and possible immunological privilege of the uterus. Short-term transplantation of trophoblast cells has been reported in guinea pigs (Borland et al. 1970) and mice (Billington 1965, Croy et al. 1984). In these studies, ectopically transplanted trophoblast cells were shown to survive for up to 20 days (Borland et al. 1970). A recent study of ectopic transplantation of trophoblast stem cells in mice demonstrated their capacity to survive for several months and to modulate their local immune environment (Epple-Farmer et al. 2009). Previous studies from our group have shown that equine chorionic girdle binucleate trophoblast cells survive for at least 28 days after transplantation in ectopic sites such as the vulvar mucosa and under the skin (Adams & Antczak 2001, de Mestre et al. 2008). At 28 days post-transplantation, the trophoblast cells were terminally differentiated and restricted to the transplant site, suggesting that they do not proliferate or migrate (Adams & Antczak 2001, de Mestre et al. 2008). It is not known how long fully allogeneic ectopically transplanted chorionic girdle trophoblast cells can survive in non-pregnant recipients. In normal horse pregnancy, chorionic girdle trophoblast cells invade the endometrium at days 36–38 of gestation and transform into equine chorionic gonadotrophin (eCG) secreting endometrial cup trophoblasts that have a 60–90 day lifespan (Allen & Moor 1972). A similar survival time for trophoblast cells located in the gravid endometrium of mares or in ectopic sites such as the vulva would suggest that immunoprotective mechanisms inherent to the trophoblast cell are of critical importance to the development of maternal-fetal tolerance.

Our previous studies also showed transplanted trophoblast cells expressed eCG, but whether the protein is secreted and functional is not known (Adams & Antczak 2001, de Mestre et al. 2008). Given the high secretory capacity of these trophoblast cells during equine pregnancy, here we hypothesize that ectopically transplanted chorionic girdle trophoblast cells may also be a source of biologically active eCG capable of modulating the reproductive physiology of non-pregnant recipients. The late attachment of the equine conceptus permits isolation of highly purified populations of chorionic girdle trophoblast cells with minimal manipulation. In addition, equine and human pregnancy are of similar gestational length, and both are characterized by abundant secretion of a trophoblast specific gonadotrophin, CG. These features make the horse an attractive species in which to assess the long-term survival potential and function of transplanted allogeneic trophoblast cells. 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 eCG can be systemically delivered by transplanted chorionic girdle cells, and (iii) if eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus.

Results

Transplanted allogeneic trophoblast cells survive and function the non-pregnant mare for up to 3 months

Following establishment in the gravid endometrium, terminally differentiated chorionic girdle trophoblast cells have a lifespan of 60 to 90 days in pregnant mares (Allen 1969). We determined the lifespan of ectopically transplanted chorionic girdle trophoblast cells in non-pregnant mares. First, survival of transplanted chorionic girdle trophoblast cells was measured by direct observation in biopsies, and then indirectly, through measurement of eCG. We transplanted allogeneic chorionic girdle trophoblast tissue from MHC typed donors into three non-pregnant immunocompetent nulliparous mares of known MHC haplotype (Table 1). Approximately 100 mg of tissue equivalent to about 20 × 106 cells was delivered via hypodermic injection into the vulvar mucosa of the recipients immediately following recovery of the trophoblast cells. To establish the fate of the transplanted cells, we performed weekly biopsies of the transplant site between weeks 3 and 8 post-transplantation. Immunohistochemical labeling of cryostat sections of the biopsies using a monoclonal antibody to equine trophoblast (102.1 (Oriol et al. 1991)) confirmed that trophoblast cells survived for at least 8 weeks (Fig. 1A). Serial sections of all biopsies tested with an isotype negative control antibody did not result in labeling of cells (representative section, Fig. 1B). Trophoblast cells were detected in sections of biopsies obtained at weeks 3 and 4 (3/3 mares), weeks 5, 6, and 7 (2/3 mares) and week 8 (1/3 mares), indicating the lifespan of the cells differed between recipients. The morphology of the trophoblast cells was consistent with terminally differentiated binucleate cells of the endometrial cups (Fig. 1C).

Table 1.

MHC haplotypes of donors and recipients of equine chorionic girdle trophoblast cells, sex of donor conceptus, and serum progesterone levels of recipients on the day of transplantation

Trophoblast Donor Sex of

Conceptus1 		Trophoblast recipient Progesterone
(ng/ml)
MHC haplotype ID MHC haplotype
Group 1: Transplantation of chorionic girdle trophoblast followed by weekly biopsies
Dam A2/A5 Male 3882 A3/?2 0.05
Sire A2/A23
Dam A2/? Male 3903 A9/A9 0.05
Sire A2/A2
Dam A3/A19 Male 3837 A10/? 0.51
Sire A3/A3
Group 2: Transplantation of chorionic girdle trophoblast without biopsies
Dam (Not A2 or A3) Male 3901 A2/A19 0.05
Sire A2/A2
Dam A2/? Female 3902 A6/? 0.07
Sire A2/A2
Dam A3/? Male 3883 A10/? 0.09
Sire A2/A2
Dam A3/A3 Male 3836 A10/? 7.5
Sire A2/A2
NT4 Unknown 3941 NT 5.1
NT Unknown 3942 NT 1.0
NT Unknown 3943 NT 0.2
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1

Determined by Y chromosome specific PCR (Supplementary Fig. 1).

2

Probable MHC heterozygote, but only a single haplotype was detected by serological testing

3

Known MHC homozygote

4

Not typed for MHC markers

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

Fig. 1

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(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.

To determine the functionality of the transplanted cells, serial sections of biopsies taken from transplant sites were also labelled with monoclonal antibodies to differentiation molecules of invasive trophoblast and eCG. Without exception, all transplanted trophoblast cells were labelled by the antibodies to invasive trophoblast (data not shown) and eCG (Fig. 2A). Serial sections of all biopsies tested with an isotype control antibody did not result in labeling of cells. To assess if eCG was secreted by the trophoblast cells and detectable in the peripheral blood of recipient mares that were biopsied, we measured serum concentrations of eCG. eCG was detected in all three recipients, with peak eCG concentrations of 3–14 iu/ml reached between 14 and 28 days post-transplantation (Fig. 2B). The highest peak concentration of eCG was measured in the mare in which trophoblast cells were detectable in all six biopsies, further supporting the use of eCG levels as an indirect measure of trophoblast cell survival.

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

Fig. 2

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(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).

We then determined the lifespan of the transplanted trophoblast cells in the absence of biopsies in a second group of non-pregnant recipient mares (n=7, Table 1), using serum eCG levels as an indirect measure of cell survival. Ultrasonographic evaluation of the ovaries of recipients, together with measurement of serum progesterone levels (Table 1), indicated that five recipient mares were in late estrus and two mares were in early diestrus at the time they received their transplants. eCG was detected in the peripheral blood of all seven recipient mares that received chorionic girdle trophoblast (Fig. 3A), with peak concentrations ranging from 5 to 25 iu/ml. Serum levels of eCG returned to day 0 concentrations between 77 and 112 days post-transplantation. Serum eCG was never detected in control mares (<0.6 iu/ml) (Fig. 4). The lifespan of the transplanted cells was extrapolated from serum eCG concentrations. The median lifespan of transplanted chorionic girdle trophoblast cells was 75 days (95% CI 55–94 days, Fig. 3B).

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

Fig. 3

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(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.

Fig. 4

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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.

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

Equine chorionic gonadotrophin has LH-like activity in the mare that promotes luteinization of follicles leading to sustained serum progesterone levels (Daels et al. 1998). To determine whether the eCG secreted by transplanted cells was biologically active, we monitored three aspects of the reproductive physiology of mares that received chorionic girdle trophoblast (n=4) or control treatments (n=4). Three of the control mares received transplants of equivalent amounts of allantochorion tissue, injected as for the chorionic girdle. The fourth control mare was injected with medium alone. Serum progesterone levels in the chorionic girdle trophoblast recipients remained elevated between 74 and 112 days post-transplantation (Fig. 4). Serum progesterone concentrations in three of the four control mares dropped below 0.6 ng/ml three times in the 66 days that followed transplantation (Fig. 4), consistent with normal estrous cyclicity. Serum progesterone in a single control mare dropped below 0.6 ng/ml at 25 days then again at 98 days consistent with a normal estrous cycle followed by prolonged diestrus.

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

The total number of luteal structures present in each mare was determined biweekly using transrectal ultrasonography of the ovaries. Chorionic girdle trophoblast recipients had a significant increase in average number of luteal structures compared to the control group (p=0.0163) (Fig. 5A). The appearance of multiple luteal structures on the ovaries of recipients of chorionic girdle trophoblast, together with the duration of progesterone production, was correlated with the appearance and subsequent disappearance of serum equine eCG (Fig. 4, Fig. 5A). We also compared the interovulatory period in recipients of chorionic girdle trophoblast and control mares. Chorionic girdle trophoblast recipients had a median interovulatory period of 84 +/−11.6 days (Fig. 5B), which was significantly prolonged when compared with the control group: 21 days +/− 7.2 days (p=0.0294). Together, these results indicate that the mares transplanted with chorionic girdle trophoblast cells showed changes in reproductive physiology consistent with the known effects of eCG in the pregnant mare.

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

Fig. 5

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(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.

Transplanted chorionic girdle trophoblast cells suppress estrus-driven behavioral changes

Approximately 4–7 days preceding ovulation and up to 2 days following ovulation, mares show estrus driven behavioral changes of receptivity that manifest dramatically when the mare is presented to a stallion. These behavioral characteristics are driven by rising estrogen levels and have been shown to be repressed by administration of exogenous progesterone (Asa et al. 1984). Chorionic girdle trophoblast recipients (n=4) and control mares (n=4) were scored biweekly for estrous behavior following exposure to a stallion, beginning seven days before transplantation. Both chorionic girdle trophoblast recipients and control mares showed classic estrous behavior at day −4 to +4 (Fig. 6). The control mares showed estrous behavioral characteristics again at days 18 to 25, and at days 32 to 39, consistent with the 17 to 22 day estrous cycle of the mare. In contrast, the chorionic girdle trophoblast recipients failed to show estrous behavior in the equivalent time period. Suppression of classic signs of estrus behavior (estrus score 2) in chorionic girdle trophoblast recipients continued until at least day 74 post-transplantation.

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

Fig. 6

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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 95th (upper error bars) and 5th (lower error bars) percentile of the data.

Materials and Methods

Horses

These studies used stallions and mares of several breeds and various ages donated or bred at Cornell University or Equibreed. Non-pregnant transplant recipients were nulliparous mares aged between 3 and 7 years. Horses were maintained at the Baker Institute for Animal Health, Cornell University, USA, or Equibreed, NZ. Animal care was performed in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of Cornell University, and Massey University Animal Ethics Committee (MUAEC). MHC class I typing of horses was determined as previously described (Adams & Antczak 2001). At Cornell University, stallions homozygous for MHC ELA-A2 or ELA-A3 haplotypes were used as semen donors; pregnancies were established as previously described (Adams & Antczak 2001).

Transplantation of chorionic girdle

Chorionic girdles from day 33–34 conceptuses were obtained non-surgically using established methods (Adams & Antczak 2001). Girdles were dissected free of allantochorion and chorion and placed into serum free medium (de Mestre et al. 2008). Following dissection, each experimental recipient was transplanted with an entire chorionic girdle from a single conceptus. Recipient mares were sedated with 0.4 mg/kg Xylazine (Lloyd Laboratories, IO) and 0.001 mg/kg butorphanol tartrate (Fort Dodge, IO). The transplant site (vulva) was prepared aseptically. Sectioned chorionic girdle strips were injected subcutaneously into the vulvar sub-mucosa of recipients in 0.5 ml of DMEM using a 1.0 ml syringe. For Group 1 recipients, where serial biopsy samples of transplant sites were obtained (n=3; Table 1), chorionic girdle trophoblast obtained from one conceptus was divided into six pieces and delivered in six independent sites in the vulva. Biopsies were obtained from the recipients at 21, 28, 35, 42, 49 and 56 days after transplantation as previously described (de Mestre et al. 2008). For Group 2 recipients (Table 1) no biopsies were performed. The seven recipients each received an entire chorionic girdle obtained from a single conceptus, injected into three sites in the vulva. As controls for Group 2, non-pregnant nulliparous mares were transplanted with a similar volume of non-invasive trophoblast tissue (allantochorion) using the same technique (n=3) or injected with medium alone (n=1). All recipients were clinically healthy at the time of transplantation and throughout the study period.

Hormone assays

The concentration of eCG in serum was determined using an enzyme linked immunoassay as previously described (de Mestre et al. 2008). The concentration of equine progesterone in serum samples was determined using a solid-phase radioimmunoassay (Reimers et al. 1991).

Tissues and immunohistochemistry

Biopsy samples of the trophoblast transplant sites were obtained and frozen as previously described (de Mestre et al. 2008) or fixed in 4% buffered formaldehyde for histology. Tissue sections of 6 µm thickness were cut and stained using indirect immunoperoxidase assays (Adams & Antczak 2001), employing monoclonal antibodies specific to all equine trophoblast cells (antibody 102.1), invasive equine trophoblast (antibody 71.3), eCG (67.1) (Oriol et al. 1991), and an isotype-matched negative control.

Calculations of trophoblast lifespan

The lifespan of transplanted trophoblast cells was determined from circulating serum eCG concentrations. Trophoblast cells were considered present if the serum concentration of eCG was greater than 1.5 iu/ml (2–3 fold increase over day 0 concentrations) and the concentration of eCG had not decreased by more than 50% over the preceding 6 days (representing the half-life of the protein).

Interovulatory period & measurement of luteal structures

Estrous cyclicity was monitored in Group 2 chorionic girdle trophoblast recipients (n=4) and control recipient mares (n=4) using transrectal ovarian and uterine ultrasonography (McKinnon 1993) performed using an Aloka SSD-500 instrument (Aloka, Wallingford, CT), and biweekly monitoring of serum progesterone concentrations. Ovulation was determined using a combination of ovarian ultrasonography and changes in serum progesterone. The total number of luteal structures was determined using transrectal ovarian ultrasonography.

Monitoring estrous behavior

Estrous behavior was determined by presenting a stallion to the mare. A score of 0 indicated no receptivity to the stallion, a score of 1 indicated partial receptivity to the stallion, and a score of 2 indicated full estrous receptivity marked by vulvar winking accompanied by urination.

Genomic DNA isolation and PCR

Y chromosome specific PCR primers were designed from the equine YH12 microsatellite sequence (Wallner et al. 2004) using Primer3 on the web (Rozen & Skaletsky 2000). Genomic DNA was isolated from frozen tissues using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA.) following the manufacturer’s protocol. PCR amplification was carried out in 25 µl reactions consisting of 75 ng genomic DNA, 1× PCR Buffer (Invitrogen), 0.2 mM each dNTP, 1.5 mM MgCl, 0.25 µM each primer, and 1.25 ul recombinant Taq DNA Polymerase (Invitrogen, Carlsbad, CA.). PCR products were loaded on a 1% agarose, electrophoresed at 75 V for 1.5 hrs, stained with Ethidium Bromide, and visualized on a transilluminator. Positive and negative controls consisting of gDNA from a known male and female horse, respectively, were included in each PCR.

Statistical analysis

Statistical analysis was performed using Graphpad Software, Prism 5 for Mac OS X Version 5.0c (Graphpad Inc, La Jolla, CA, USA). Data was assessed for normal distribution using D’Agostino and Pearson normality test. In the situation that data sets were too small to perform this test, then a non-Gaussian distribution was assumed. The number of luteal structures between control (n=4) and trophoblast recipient (n=4) mares was compared and analyzed using a 2 way ANOVA with Bonferroni’s correction and an alpha level of 5%. The differences in interovulatory period between control (n=4) and trophoblast recipient (n=4) mares were analyzed using a Mann-Whitney test, with an alpha level of 5%.

DISCUSSION

The studies reported here demonstrate that fully allogeneic trophoblast cells transplanted to ectopic sites can survive and avoid immune destruction for up to 105 days while producing easily detectable levels of their primary secreted product, eCG. Consistent with our hypothesis, the eCG secreted by the cells was biologically active and suppressed behavioral signs associated with estrus.

The survival of equine trophoblast allografts for over 3 months is significantly prolonged compared to that reported for other tissues, including conventional skin allografts in the horse (Adams et al., 2007). The mechanisms responsible for the avoidance of cell destruction are not fully understood, although immunosuppressive factors expressed and/or secreted by trophoblast cells appear to strongly influence their fate. There are limited reports of allogeneic trophoblast transplantation in other species. Mid to late gestation guinea pig trophoblast cells injected intradermally were reported to survive for 14–20 days (Borland et al. 1970). The shorter survival period of allogeneic trophoblast cells in guinea pigs may be related to a number of factors, including the use of trophoblast cells obtained late in the gestation period, the method used to isolate the cells, the site of transplantation, or the short gestation period of guinea pigs compared to that of horse mares. Other studies show the site of injection influences the outcome of the transplants. Transplants of murine trophoblast stem cells fail when injected subcutaneously or into the tail vein of mice (Erlebacher et al. 2002, Kibschull et al. 2004), but survive for up to 3–4 months when injected directly into the portal vein and lodge in the liver (Epple-Farmer et al. 2009). The latter is a similar lifespan to our observations here of trophoblast transplanted to the vulva. The eventual demise of the equine trophoblast grafts reported here by 105 days fits well with the 60 to 90 day lifespan of the endometrial cups in normal horse pregnancy (Allen 1969). It would be interesting to determine if a period of cell culture could further extend the survival period of trophoblast cells, as has been shown for thyroid tissue (Lafferty et al. 1975).

The pregnant state in mammals alters many aspects of the physiology of the mother, including the immune system, where a multitude of changes have been described in both local and peripheral immunity (Moffett & Loke 2006, Seavey & Mosmann 2008). What remains unresolved is how important each of these changes is for the outcome of the pregnancy. This problem has been illustrated by several studies that used gene deletion in mice (Trowsdale & Betz 2006), which often show normal pregnancy progression despite lack of expression of the deleted molecule in the placenta or decidua. It is often difficult even to resolve the relative importance of contributions from the uterine environment, the hormonal milieu of pregnancy, or the innate defense mechanisms of the trophoblast. Using a model of transplantation of trophoblast into non-pregnant animals, we have demonstrated the importance of mechanisms inherent to the trophoblast cells for their survival. Furthermore, the similar lifespan of the equine trophoblast cells in the pregnant and non-pregnant states (Allen & Moor 1972) suggests that molecules specific to the uterine environment are not critical to the survival of fetally derived trophoblast cells. Our studies cannot rule out a role for maternal hormones, such as progesterone, in modulation of important mediators of immunity during pregnancy. In the case of equine pregnancy, however, the trophoblast cells themselves appear to be the “master regulators” of their own fate.

The transplanted equine trophoblast cells differentiated into mature, binucleate endometrial cup trophoblasts that produced sufficient amounts of CG to alter the reproductive physiology and behavior of the transplant recipients. This is remarkable, given the very small mass of the transplants. In normal horse pregnancy, the endometrial cups are easily observed on the lumenal surface of the uterus, and about 10 grams of endometrial cup tissue can be recovered (de Mestre 2010). In contrast, the trophoblast transplants from an entire chorionic girdle cannot be seen macroscopically, and the entire mass of transplanted trophoblast tissue is less than one gram. This highlights the extraordinary strength of the CG promoters and the very high rate of secretion of CG into the tissues by the transplanted cells. CG is a heterodimeric glycoprotein comprised of alpha and beta subunits encoded by CGA and CGB genes. The strength of the equine CGA and CGB promoters differs by approximately 10 fold (de Mestre et al. 2009), a finding consistent with observations in human pregnancy (Cole 2009). This biological variation in promoter strength could be exploited to regulate the concentration of exogenously delivered molecules by stably transfected trophoblast cells.

Peak serum levels of eCG (5–25 iu/ml) in mares receiving transplants was lower than that reported for pregnant mares (50–200 iu/ml) but much higher than the levels achieved in mares administrated exogenous eCG via injection (Dinger et al. 1982, Daels et al. 1998). The presence of serum eCG in non-pregnant transplanted mares correlated with the formation of secondary luteal structures and luteal steroidgenesis, consistent with the bioactivity of eCG reported in pregnant mares (Daels et al. 1998). The peak levels of progesterone fluctuated corresponding to the changes in the number of luteal structures in individual recipients, suggesting that the primary corpus luteum, as well as secondary luteal structures, were capable of luteal steroidogenesis. Human CG administered to non-pregnant mares in early diestrus has also been shown to result in increased progestin production (Watson et al., 1995). Luteal LH/CG receptors are expressed in early diestrus in the mare (Stewart & Allen 1979, Roser & Evans 1983). Collectively, our findings indicate these receptors are not only expressed, but are also capable of binding eCG during prolonged diestrus in the mare.

The unexpectedly long survival and function of ectopically transplanted trophoblast in non-immunosuppressed fully allogeneic recipients suggests several possible practical applications. It is likely that the changes in estrus behavior in the recipient mares was caused by sustained delivery of low levels of eCG. This form of cellular therapy may find utility as a means of extended suppression of unwanted estrus behavior in performance sport horses. Further in the future, there may be a role for genetically modified trophoblast cells as vehicles to deliver a wide variety of biologically active molecules.

Supplementary Material

SupplData

Sex of donor invasive trophoblast cells was determined using Y chromosome PCR. Genomic DNA (gDNA) was isolated from the fetuses that matched the donor invasive trophoblast cells, or peripheral blood mononuclear cells harvested from a stallion or mare (as controls). NTC indicates no template control. See Materials and Methods for details of assay.

Acknowledgements

We thank Leela Noronha, Lee Morris, Emily Silvela and Meleana Hinchman for assistance with animal work, and Sandra Wilsher for assistance with equine CG assays. We thank AHDC Endocrinology Lab, College of Veterinary Medicine, Cornell University, for performing progesterone assays.

Funding

This research was funded by NIH grant R01-HD049545 and the Zweig Memorial Fund. DFA is an Investigator of the Dorothy Russell Havemeyer Foundation, Inc.

Footnotes

Declarations of interest

There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SupplData

Sex of donor invasive trophoblast cells was determined using Y chromosome PCR. Genomic DNA (gDNA) was isolated from the fetuses that matched the donor invasive trophoblast cells, or peripheral blood mononuclear cells harvested from a stallion or mare (as controls). NTC indicates no template control. See Materials and Methods for details of assay.

NIHMS518968-supplement-SupplData.jpg (63.9KB, jpg)

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