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J. A. Carnegie

Summary. Rats were pretreated with oestradiol-17β or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h. Similarly, PMSG-cells secreted 16-fold more 20α-dihydroprogesterone than did E2-cells, in the absence of FSH, and 6- to 10-fold more of the 20α-reduced metabolite in the presence of FSH. In contrast, fibronectin secretion by PMSG-cells was only about 60% and 30% that by E2-cells at 24 and 48 h, respectively. Fibronectin secretion by E2- and PMSG-cells was reduced in the presence of FSH at 24 and at 48 h of culture. While cells in all treatment groups underwent spreading during culture to become elongated and irregular in outline, elevated fibronectin secretion in vitro was accompanied by enhanced cellular spreading. At 24 and 48 h of culture respectively, the mean area occupied by E2-cells on the culture surface was × 2 and × 1·6 that by PMSG-cells. Coincident with its inhibitory effect on fibronectin secretion, FSH reduced the mean area occupied by E2- and PMSG-cells on the culture surface at both time intervals. These findings suggest that granulosa cell fibronectin secretion is a feature of early follicular development. It may be that the secretion of this adhesive glycoprotein by granulosa cells provides a pool of fibronectin which is used for basement membrane deposition during follicular growth.

Keywords: fibronectin; granulosa cell; extracellular matrix; follicular development; rat

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J. A. Carnegie

Summary. Blastocysts (∼ 50 per female) were collected on Day 5 of gestation from immature Sprague–Dawley rats superovulated using FSH/hCG-loaded mini-osmotic pumps and a single injection of the LHRH analogue, des-gly10 (d-ala6)-LHRH-ethylamide. The cytoplasmic distribution of fibronectin and laminin was determined by immunofluorescence within these blastocysts, either immediately following their isolation or after they had been cultured in serum-free medium for 48–96 h (to allow trophectodermal cell attachment and outgrowth). In addition, inner cell masses (ICMs; isolated by immunosurgery) were cultured under serum-free conditions and immunofluorescently stained for the presence of the two adhesive glycoproteins. Within the freshly isolated blastocysts, positive immunostaining was obtained only for fibronectin and this was associated with the trophectodermal layer. After 48–96 h of culture, the cytoplasm of all trophectodermal cells contained both fibronectin (organized as a slightly granular network) and laminin (the staining pattern was distinctly punctate and perinuclear concentrations of immunoreactivity were evident). ICM-cells stained intensely for the presence of laminin at 48, 72 and 96 h of culture, but appeared to contain little to no fibronectin. While further studies using serum-free culture are needed to define the hormonal regulation of this process, these findings support a role for early gestation rat trophectodermal cells, in addition to the established involvement of ICM-derived parietal endodermal cells, in the synthesis of extracellular matrix components found in Reichert's membrane. The appearance of trophectoderm-associated fibronectin in freshly isolated blastocysts before the establishment of the parietal endoderm layer may implicate this glycoprotein in the provision of a substrate for the migration of these cells as they form an endodermal lining to the blastocoele.

Keywords: fibronectin; laminin; blastocyst; trophectoderm; inner cell mass; extracellular matrix

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J. A. Carnegie, J. J. Morgan, N. McDiarmid and R. Durnford

A co-culture system for bovine embryos using mitomycin-treated Vero cells and serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hatched blastocysts. In this system, it has been suggested that one contribution made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns about exposure of early embryos to serum due to its incompatibility with embryo cryosurvival. In this study, the influence of two protein supplements (synthetic serum substitute (SSS), a lipid-free human serum-derived product) and oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embryo cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal bovine serum (FBS) (recommended maintenance medium for this cell line) secreted detectable amounts of LIF (13.1 ± 0.9 pg LIF per 105 cells). Culture in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 ± 6.2 pg LIF per 105 cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v) SSS. Results of a second series of experiments in which supplementation with each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0.05) in cumulative LIF secretion with every 24 h of culture in mSOF + either SSS or ECS. In terms of embryo development and post-cryopreservation viability, medium supplementation with 2% (v/v) SSS alone versus the two-step system of 2% (v/v) SSS (days 1–4) + 10% (v/v) ECS (days 4–10) had no influence (P > 0.05) on the ability of bovine blastocysts to hatch, with or without intervening cryostorage. However, the rate of blastocyst formation (expressed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added beginning on day 4 of co-culture. In summary, Vero cell LIF secretion was increased markedly by ECS. A two-step system of medium supplementation, in which embryos are exposed to ECS beginning on day 4 of in vitro development combined high rates of blastocyst formation with cryotolerance. This effect may be a result of limiting embryo exposure to serum-derived lipid until after the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.

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B. K. Tsang, M. Li and J. A. Carnegie

Summary. The secretion of progesterone and 20α-hydroxypregn-4-en-3-one (20α-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B. These findings are consistent with the concept that microfilaments influence cell shape and steroidogenesis in granulosa cells in vitro and that FSH alters microfilament distribution and shape of cultured granulosa cells in eliciting its steroidogenic influence.

Keywords: microfilaments; steroidogenesis; FSH; granulosa cells

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Jacqueline A. Carnegie, J. S. D. Chan, M. E. McCully, H. A. Robertson and H. G. Friesen

Summary. Sections of Days 12 to 55 sheep chorion, embedded at low temperature in glycol methacrylate, were exposed to rabbit anti-ovine chorionic somatomammotrophin (oCS) to ascertain the distribution of oCS. This hormone was first detectable on Day 14, when the cytoplasm of each chorionic cell displayed a low level of fluorescence surrounding droplets which were shown to be lipid containing. At this time, all chorionic cells were uninucleate. By Day 28, binucleate chorionic cells had appeared but showed no binding of the oCS antiserum which was confined to a significant proportion of the uninucleate chorionic cells surrounding lipid droplets, as at Day 14. The same pattern of hormone distribution, although with reduced fluorescence intensity, was observed on Day 55; fluorescence indicative of antibody binding was seen only in some of the uninucleate chorionic cells. Hence, oCS was detected in chorionic tissue before the differentiation of binucleate cells (Day 14) and, at all stages, it was confined to the cytoplasm of specific uninucleate chorionic cells in close association with lipid droplets.