Requirement of inner cell mass for efficient chorionic gonadotrophin secretion by blastocysts of common marmosets (Callithrix jacchus) P. M. Summers, C. T. Taylor and M. W. Miller MRC/AFRC Comparative Physiology Research Group, Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK The role of the inner cell mass in the induction of chorionic gonadotrophin synthesis and secretion by the trophoblast of the peri-implantation primate blastocyst was studied in common marmoset monkeys. An in vitro system for the culture of blastocysts commencing with blastocysts collected 8 days after conception was developed. Chorionic gonadotrophin measured in the spent culture fluid was first detected in most blastocysts after 3 or 4 days (day 11 or 12) of culture at a time equivalent to implantation in vitro. Initial secretion of chorionic gonadotrophin coincided with development of parietal endoderm and histological appearance of syncytiotrophoblast in the polar trophoblast. Little chorionic gonadotrophin was secreted by blastocysts with a poorly developed, or absent, inner cell mass. Mural trophoblast removed from blastocysts after 2 days of culture (day 10) grew in vitro as a unilaminar vesicle but failed to secrete significant amounts of chorionic gonadotrophin. However, mural trophoblast from older blastocysts (days 13 and 14) after chorionic gonado- trophin secretion had commenced continued to secrete chorionic gonadotrophin, with trophoblast from day 14 blastocysts secreting significantly more than that from day 13. It was concluded from these studies that while mural trophoblast from marmoset blastocysts will proliferate in vitro in the absence of an inner cell mass, efficient induction of chorionic gonadotrophin secretion requires the presence of the inner cell mass or its derivatives. Once chorionic gonadotrophin secretion has commenced, secretion will continue in the absence of the inner cell mass. Introduction Little is known about the cellular and molecular mechanisms that lead to the initiation of chorionic gonadotrophin synthesis and the of the The establishment of mammalian pregnancy depends on the secretion by trophoblast primate blastocyst secretion of specific proteins by the conceptus (Roberts et al, despite the considerable progress made in the definition of chorionic 1990). Some of these proteins act directly or indirectly on the human gonadotrophin genes (Stewart et al, 1988). In corpus luteum to maintain the secretion of progesterone, which addition to leading to a better understanding of infertility in these cellular and in turn ensures an appropriate uterine milieu for the continued women, improved knowledge of molecular survival of the conceptus. This physiological interaction mechanisms could have a practical application in the development of between the conceptus and maternal tissues is referred to as the new methods of contragestation. maternal recognition of pregnancy (Short, 1969). In primates, The purpose of this study was to define the in vitro growth the product synthesized and secreted by the trophoblast of the and chorionic gonadotrophin secretion by peri-implantation implanting blastocyst that rescues the corpus luteum is the blastocysts from the common marmoset monkey (Callithrix glycoprotein, chorionic gonadotrophin (CG) (Heap et al, 1979). jacchus) and to examine the role of the inner cell mass in the the and chorionic Human fertility is widely recognized as being low (Biggers, control of growth gonadotrophin secretion 1981) and studies have shown that considerable embryonic loss by trophoblast cells. occurs during the 3 weeks following conception (Chartier et al, 1979; Miller et al, 1980; Whittaker et al, 1983). Irregular and significantly slower doubling time of peripheral blood concen¬ Materials and Methods trations of chorionic gonadotrophin have been associated with this embryonic loss (Edmonds et al, 1982; Lenton et al, 1982; Animals Lenton and Woodward, 1988), which implies that inadequate chorionic gonadotrophin secretion may be a factor contributing Marmoset monkeys were from a self-sustaining breeding to the failure of pregnancy. colony maintained at the Institute of Zoology. Embryo donors were housed in with a fertile male and 'Present address: Graduate School of Tropical Veterinary Science and Agriculture, family groups usually James Cook University, Townsville 4811 Australia. two prepubertal juveniles, and their ovarian cycles were con¬ Received 15 January 1992. trolled by the intramuscular administration of the prostaglandin Downloaded from Bioscientifica.com at 10/02/2021 07:04:15PM via free access analogue, cloprostenol (Estrumate: Coopers Animal Health Ltd, Chorionic gonadotrophin assay Crewe) during the mid- to late luteal phase (Summers et al, The of chorionic 1985). Blood samples were taken from the femoral vein 9, 11 biological activity gonadotrophin secreted and 13 days after cloprostenol injection and the day of ovula¬ into the culture medium by blastocysts or isolated trophoblast was measured in an in vitro based on the of tion (day 0) was determined by measuring plasma progesterone bioassay production testosterone mouse cells Damme et concentrations using a radioimmunoassay (Summers et al, by dispersed Leydig (Van 1985). al, 1974). Details of the method and its validation for the marmoset have been described previously (Hearn et al, 1988). Assays of samples from each blastocyst included one or two Blastocyst collection and culture control culture medium samples; concentrations detected in control culture media were subtracted from the test samples. were collected 8 Blastocysts surgically from the uterus days The sensitivity of the assay based on 90% was within after binding ovulation (Summers et al, 1987a). Individual blastocysts the range 2.3-13.6 miu ml-1. The intra- and interassay coef¬ were cultured in 200 Hams FIO medium in 96-well flat- µ ficients of variation of a marmoset plasma pool were 19.9% and bottom plastic culture trays (Nunc, Hamstrup, Denmark) at 6.91%, respectively. 37.5°C in a humidified atmosphere of 5% C02 and 95% air. The culture medium was prepared from Hams FIO powder contain¬ Histology ing glutamine (Flow Laboratories, Irvine) using twice glass distilled water; 1.2 g sodium bicarbonate 1_I was added and the Blastocysts and trophoblast vesicles were fixed in 2% pH adjusted to 7.2. The medium was supplemented with 20% glutaraldehyde in 0.05 mol phosphate buffer 1_I pH 7.4, (v/v) heat inactivated fetal calf serum (Flow Labs), penicillin G embedded in 2% agar in phosphate-buffered saline (PBS) for (8 mg in 100 ml) and streptomycin (5 mg in 100 ml) (Sigma, ease of handling, dehydrated through graded alcohols and Poole). embedded via propylene oxide in epon—araldite. The resin Blastocysts were examined each day under phase contrast block was oriented so that sections (1.5-2.0 µ ) could be cut microscopy, their stage of development and diameter (including perpendicular to the inner cell mass. Sections were cut until the zona pellucida) recorded and they were then transferred into a middle of the inner cell mass was reached and were stained with fresh culture well. The extent of parietal endoderm develop¬ 1% toluidine blue. ment was estimated by rotating the blastocyst in the culture medium while were examined When they microscopically. Immunosurgical isolation of the inner cell mass blastocysts formed a firm trophoblast attachment and out¬ The inner cell mass was isolated from 10 growth on the bottom of the culture well, the medium was day blastocysts removed carefully and replaced with fresh medium without after the herniated trophoblast cells were removed and the disturbing the blastocyst. Spent culture medium and that of the blastocysts cultured for 3—5 h to allow the blastocoel to reform. zona was brief control wells was stored at 20°C until assayed for chorionic The pellucida removed by exposure (5-10 s) to gonadotrophin concentration.— acidic (pH 2.5) Tyrode's solution (Nicholson et al, 1975) and the zona-free blastocyst incubated for 30 min at 37°C in rabbit anti- marmoset antisera diluted 1:64 in Ham's FIO plus 20% fetal calf Removal of mural trophoblast (trophecloderm) cells serum (FCS). Anti-marmoset antisera were produced in a New Zealand white rabbit by an initial i.v. injection of 3.2 107 were to a of Day 8 blastocysts subjected series micromanipu- marmoset spleen cells followed by three i.v. injections of lative with the aid of a Leitz procedures micromanipulator 8-16 106 peripheral blood leucocytes at 10 day intervals. (Summers et al, 1988) to obtain mural assembly trophoblast. Blood samples were taken from the rabbit 10 days after the last a was in a of Briefly, blastocyst placed drop Dulbecco's phosphate- injection; the serum heat treated at 56°C for 30 min and stored buffered saline on a glass depression slide, its embryonic pole at -20°C. held a zona the by suction pipette and the pellucida opposite After incubation in anti-marmoset antisera, the blastocyst inner cell mass with two microhooks. The quickly opened was washed three times in fresh Ham's FIO plus 20% FCS and blastocyst was cultured for 2 to as 10 days (referred day then incubated in fresh guinea-pig serum diluted 1:16 in Ham's blastocysts) by which time some of the mural trophoblast had FIO plus 20% FCS for 30 min at 37°C. The lysed trophoblast herniated the break made in the zona The through pellucida. cells were then removed by gentle pipetting. The isolated inner herniated which contained 30-50 cells (Summers et trophoblast cell mass was