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Tumor Necrosis Factor Decreases the Viability of Mouse Blastocysts In Vitro and In Vivo¹

^a University of Louvain Medical School, Physiology of Human Reproduction Research Unit, B-1200 Brussels, Belgium ^b Pig Research Institute, Department of Applied Biology, Miaoli, Taiwan

	ABSTRACT

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Mouse blastocysts were exposed for 24 h to various concentrations of recombinant mouse tumor necrosis factor (TNF) and observed for their capacity to implant in vitro on a fibronectin-coated substrate or to develop in vivo after their transfer into surrogate females. Compared with findings in control blastocysts, exposure to TNF resulted in a significant reduction in the average number of cells in the inner cell mass (ICM) lineage. This effect was associated with a significant increase in the frequency of cells identified as engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling technique. No difference was found in the incidence of nuclear fragmentation between control and TNF-exposed blastocysts. When TNF-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to maintain a structured ICM cluster at the center of the trophectoderm outgrowth. Although no difference was found in the average surface area of the outgrowths, implants derived from TNF-treated blastocysts contained significantly fewer nuclei than implants from control embryos. After transfer into recipient mice, TNF-pretreated blastocysts implanted at about the same rate as control embryos, but a significantly higher rate of resorption was found among fetuses after exposure to the cytokine. In addition, the weight of the surviving fetuses was significantly lower than for control fetuses. These data indicate that the impact of TNF on blastocysts is specifically aimed at the ICM lineage and that TNF decreases the ability of embryos to differentiate into fetuses after implantation.

	INTRODUCTION

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There is growing evidence indicative of the participation of a large array of cytokines in the regulation of both embryo development and uterine decidualization at the time of implantation [1]. Tumor necrosis factor (TNF) is one of the cytokines whose role is extensively studied in the context of the interactions between the implanting embryo and the receptive uterus [2]. In rodents, TNF synthesis has been detected in the uterus at the time of implantation [3], and the presence of TNF receptors has been demonstrated in blastocysts [4], in trophectoderm (TE) cells [5], and in embryonic stem (ES) cells [6, 7]. In vitro experiments have shown that TNF decreases the rate of cell proliferation in the inner cell mass (ICM) lineage of blastocysts [8], the ability of ES cells to differentiate [7], and the expression level of receptors for other cytokines in the TE lineage [5]. These observations strongly suggest that the influence of TNF on implanting embryos is predominantly inhibitory and that the ICM and TE lineages present cell-specific response patterns to the cytokine. Recent investigations have shown that the production of TNF is up-regulated in the reproductive tract of the diabetic pregnant rat [9, 10], suggesting that embryos are at risk of being exposed to excess concentrations of the cytokine at the time of implantation in the diabetic uterus. In the present study, we investigated whether incubating mouse embryos with TNF during the blastocyst phase results in a decreased ability to develop beyond implantation in vitro and in vivo.

	MATERIALS AND METHODS

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Preimplantation Development

Blastocysts were recovered from spontaneously ovulating NMRI mice on Day 4 of pregnancy; the blastocysts were randomized and incubated for 24 h in Earle's Balanced Saline Solution (Gibco, Paisley, UK) supplemented with 0.3% BSA, 1 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO₂. Incubations were performed in the absence or presence (5 or 50 ng/ml) of mouse recombinant TNF in groups of 8–10 embryos per 500 µl of culture medium. The average morphological score of the blastocysts was assessed before and after incubation according to a developmental scale described elsewhere [4]. Blastocysts were also examined for the number of cells in the ICM and TE lineages, using a differential staining technique that discriminates the two cellular lineages as described previously [4]. Careful analysis of the stained blastocysts allowed for the detection of cells whose nucleus was fragmented (karyorrhexis). Nuclear fragmentation was characterized by a pattern of highly fluorescent DNA particles still contained within intact cell boundaries. Nuclei with degraded chromatin (karyolysis) were detected in blastocysts using a cell-death detection technique based on the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) principle [11], which was applied to embryos according to a protocol described elsewhere [8].

Periimplantation Development

Control and TNF-treated blastocysts were transferred onto fibronectin-coated culture wells in 100 µl of CMRL-1066 medium (Gibco) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin for 48 h. The embryos were then classified according to the morphological aspect of the ICM and TE structures as described previously [12]. The surface area of the TE layer was also measured in outgrown blastocysts after photography. To count the total number of cells per embryo, outgrowths were treated in 30 µl of 0.5% sodium citrate; they were then dried under partial vacuum at 55°C for 30–45 min and treated with 100 µl of fixative solution at room temperature for 30 min [12]. Cells were counted by staining the embryos in 0.4% Giemsa. Control and TNF-treated blastocysts were also transferred into surrogate mice. The recipients were prepared by mating (NMRI x B6D2F1) F1 females with vasectomized NMRI males and were used on pseudopregnancy Day 3. In each recipient, 5–8 control blastocysts were placed in one uterine horn, and an equal number of TNF-treated embryos were introduced into the other uterine horn. The surrogate mice were analyzed 11 days later for the frequencies of resorptions and surviving fetuses. Fetal and placental wet weights were measured immediately after dissection.

	RESULTS

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Exposure to TNF

Embryos were collected on Day 4 of pregnancy and incubated for 24 h in the presence or absence of TNF. At the start of the incubation period, the embryos were at the early-medium blastocyst stage (mean morphological score was 7.82 ± 0.10) and contained an average of 37.22 ± 1.57 cells (27 embryos). Blastocysts exposed to 5 or 50 ng/ml of TNF developed less well (p = 0.05, Fig. 1A) during the incubation period and were found to contain fewer cells after 24 h (p = 0.05, Fig. 1B) than control blastocysts. The differential staining technique revealed that the effect of TNF on cell number was more pronounced in the ICM than in the TE. A 17% cell deficiency was observed in the ICM lineage following exposure to 50 ng/ml of TNF. Close examination of the differentially stained blastocysts showed that the incidence of nuclear fragmentation (karyorrhexis) was not increased by TNF (p = 0.38). Control frequencies of karyorrhexis were 1.05 ± 0.45% in the ICM and 0.89 ± 0.34% in the TE at the beginning of the incubation time and were 1.85 ± 0.36% and 0.73 ± 0.17% after 24 h. Application of the TUNEL technique coupled with bisbenzimide staining confirmed the absence of effect on karyorrhexis (p = 0.77), but demonstrated that exposure to TNF resulted in a dose-dependent increase in the frequency of nuclei displaying signs of chromatin degradation (karyolysis) (p = 0.01, Fig. 1C).

View larger version (33K): [in this window] [in a new window]   FIG. 1. Impact of TNF on blastocyst development. A) Morphological score of blastocysts exposed for 24 h to increasing concentrations of TNF. B) Total number of cells per blastocyst (TOT) and differential cell numbers in the ICM and TE lineages. C) Frequencies of nuclei labeled as fragmented (FR) or with degraded chromatin (TUNEL-positive, TN) as percentages of the total number of cells per blastocyst. Data are given as means ± SE and based on at least 54 values per group in A, 47 values in B, and 22 values in C, pooled from at least five reproducible experiments. * and ** represent statistical difference at p = 0.05 and p = 0.01, respectively, from the control mean value by one-way ANOVA. The scale of the ordinate in A has been enlarged.

Development In Vitro

In order to determine whether a 24-h exposure to TNF would impair their subsequent growth in vitro, blastocysts were incubated with 50 ng/ml of the cytokine and then transferred (in the absence of TNF) onto fibronectin-coated dishes for 48 h. At the end of the incubation period, 99% of the control blastocysts had attached and outgrown compared with 95% in the TNF-pretreated group. Compared with control outgrowths, fewer implants derived from TNF-pretreated blastocysts featured a compact and structured ICM at the center of the TE layer, and more of them showed an apparently normal TE expansion surrounding a limited cluster of ICM cells (p = 0.05, Fig. 2A). Measurement of surface area indicated that TNF pretreatment had no consequence on TE spreading (55905 ± 16662 µm² versus the control value of 59404 ± 13813 µm², p = 0.68). Further analysis of the outgrowths, however, showed that the number of nuclei per outgrowth was decreased after TNF pretreatment (p = 0.05, Fig. 2B). When the TUNEL-bisbenzimide staining method was used, neither the incidence of karyorrhexis (p = 0.53) nor the incidence of karyolysis (p = 0.44) per outgrowth was found to be influenced by the TNF pretreatment (Fig. 2C).

View larger version (29K): [in this window] [in a new window]   FIG. 2. Impact of TNF pretreatment on development in vitro. A) Morphological distribution of embryos after preexposure to 50 ng/ml TNF (TNF) or not (control, CTR) according to whether they have attached to the substrate (ATT) or developed into outgrowths. Outgrowths were classified based on the observation of a compact ICM containing both primitive endoderm and ectoderm lineages (ICM+++), a destructured ICM cluster (ICM++), or only a group of scattered ICM cells (ICM+) at the center of the TE layer. B) Number of nuclei per outgrowth. C) Frequencies of nuclei labeled as fragmented (FR) or with degraded chromatin (TN) per outgrowth. Results were based on at least 103 values per group in A, 45 values in B, and 32 values in C, pooled from at least five reproducible experiments. Data in A were tested by chi-square analysis and showed a statistical difference between control and TNF-pretreated groups at p = 0.05. Data in B and C were analyzed by Student's t-test with * indicating a statistical difference at p = 0.05 from the control mean value.

Development In Vivo

The impact of TNF on postimplantation growth in vivo was addressed by exposing blastocysts to 0 ng/ml or 50 ng/ml of the cytokine for 24 h and transferring them in parallel into pairs of uterine horns in Day 4 pseudopregnant mice. Assessment of the uterine content was performed 11 days after transfer. Control blastocysts (117 of 190 embryos in 25 recipients) implanted at a rate of about 62% (Fig. 3A), and TNF pretreatment induced a 17% decrease in that proportion (98 of 191 embryos) (p = 0.16). In contrast, the proportion of implanted embryos that failed to develop normally was 39% higher in the TNF-pretreated group (70 of 98 implants) in comparison to the control group (60 of 117 implants) (p = 0.05, Fig. 3B). Resorbed fetuses resembled small dark moles without distinct structures. Although the external examination of surviving fetuses did not reveal any gross morphological alteration, a comparison of fetal weight indicated a 15% decrease in the fetuses derived from TNF-pretreated blastocysts (p = 0.05, Fig. 3C). No difference was found in placental weights between the two groups (p = 0.68).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Impact of TNF pretreatment on development in vivo. A) Implantation rate and B) resorption rate following the transfer of control (CTR) or TNF-pretreated (TNF) blastocysts into surrogate mice and uterine examination 11 days later. C) Fetal and placental weights in surviving fetuses. Data were based on the intrauterine transfer of 190 control embryos and 191 TNF-pretreated blastocysts and were analyzed by Student's t-test, with * indicating a statistical difference at p = 0.05 from the control mean value.

	DISCUSSION

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The present investigation on spontaneously ovulated NMRI blastocysts corroborates several of the developmental alterations that have been described in blastocysts from superovulated OF-1 mice upon their exposure to TNF, i.e., delayed morphological progression and decreased cell proliferation in the absence of detectable nuclear fragmentation [4]. In both embryo strains, the cellular deficit induced by TNF was predominantly aimed at the ICM lineage. In addition to using the incidence of nuclear fragmentation (karyorrhexis) as a marker of cell death, in the present study we exploited the TUNEL staining technique to assess the occurrence of nuclear chromatin degradation (karyolysis). In accordance with earlier observations on rat blastocysts [8] and mouse ES cells [7], exposing mouse blastocysts to TNF resulted in an increase in the frequency of karyolysis without simultaneous change in karyorrhexis. The reason underlying this discrepancy may be related to the consideration that karyolysis and karyorrhexis require the activation of distinct sets of destructive enzymes: endonucleases for internucleosomal DNA cleavage during karyolysis and proteases targeting the nuclear envelope during karyorrhexis. Experiments will have to be performed to determine whether the apoptotic signal cascade triggered by the binding of TNF to p60 TNF receptors, which are the only receptor type expressed for this cytokine at the cell surface of mouse blastocysts [4], is specific for the stimulation of karyolytic enzymes in these embryos.

The viability of TNF-exposed blastocysts was analyzed after transfer into fibronectin-coated dishes. Compared with the control group, fewer TNF-pretreated blastocysts were able to develop beyond attachment and to maintain a central ICM cluster featuring both primitive endoderm and ectoderm cell layers. Although the average surface area of the outgrowths was not different, those implants derived from TNF-pretreated blastocysts were found to contain fewer nuclei. Experiments based on the incorporation of 5-bromo-2'-deoxyuridine into outgrowths from TNF-pretreated embryos (unpublished results) confirmed the deficit in cell number per implant. Because these outgrowths do not present signs of persisting occurrence of cell death, it is assumed that the cell number difference is secondary to the irreversible loss of karyolytic cells detected immediately after TNF treatment. Our observations also indicate that the consequences of a transient exposure to TNF before attachment are more severe than those induced by the continuous presence of TNF throughout the outgrowing process [13]. Consonant with these results in vitro, data obtained after embryo transfer reinforce the concept that cells of the ICM lineage are more sensitive than TE cells. Resorption rates and fetal weights were found to be altered whereas implantation rates and placental weights were not affected. Previous studies have demonstrated that two effects of TNF are the inhibition of cell proliferation in the ICM lineage [4] and the alteration of the differentiation potential of ES cells [7]. These effects probably contribute to the morphogenetical events leading to the high frequency of fetal resorption following TNF pretreatment. Our investigation is continuing into description of the anomalies afflicting the resorbed fetuses.

Previous experiments using stimulating cytokines and growth factors to which embryos are likely to be exposed in the uterine milieu have shown a correlation between their positive impact on blastocyst development in vitro and enhanced posttransfer outcome. For instance, epidermal growth factor has been reported to stimulate outgrowth proliferation in culture [14] and to increase the implantation rate after transfer [15] in mice, while insulin has been found to stimulate cell proliferation in the ICM in vitro [16] and to increase the posttransfer rates of implantation and fetal survival [17] in rats. Similarly, colony-stimulating factor-1 has been shown to stimulate outgrowth expansion in vitro [18] and to increase the proportion of embryos that develop normally after transfer [19] in the mouse. The present study confirms that in contrast to these embryotrophic effectors, TNF should be viewed as a negative determinant of early development. While TNF is certainly beneficial to the regulation of blastocyst growth and implantation if secreted at the correct time, location, and concentration, it is evident that its anti-proliferative and anti-differentiative action would prove extremely deleterious to the embryo when it is overproduced by the cells lining the reproductive tract. Although the local concentration of TNF within the uterine lumen is still unknown, enhanced TNF synthesis has been described in the uterus of pregnant diabetic rats [9, 10] and in cases of stress-triggered [20] and spontaneous [21] abortions in mice. In a preceding report, we showed that the serum level of TNF in the diabetic pregnant rat was about 5 ng/ml [10], which is close to the K_d of the high affinity of the p60 TNF receptor [22, 23]. TNF being produced locally by the epithelial cells of the uterus, it is likely that the concentration of the cytokine in the vicinity of the blastocyst might be higher than the serum level, and in the range of the concentrations used in this study. Further investigation into the (dys)-regulation of uterine TNF production and into its influence on embryo growth would provide us with an important key to understanding the many aspects of early pregnancy failure.

In conclusion, the present study clearly demonstrates that exposing mouse blastocysts to TNF for 24 h results in 1) an increased incidence of karyolysis and a decreased rate of cell proliferation in the ICM lineage after treatment, 2) a decreased ability to adhere to a fibronectin-coated substrate and to maintain a structured ICM cluster following TE spreading, and 3) an increased rate of fetal resorption after transfer into pseudopregnant females.

	FOOTNOTES

 
¹ This work was supported by a grant from the Juvenile Diabetes Foundation International and by grants from the Fonds de la Recherche Scientifique Médicale and the Fonds National de la Recherche Scientifique of Belgium. S.P. is Chercheur Qualifié FNRS.

² Correspondence: R. De Hertogh, OBST 5330 Research Unit, University of Louvain Medical School, 53 Avenue Mounier, B-1200 Brussels, Belgium. FAX: 32 2 7645396; dehertogh{at}obst.ucl.ac.be

Accepted: September 29, 1998.

Received: July 7, 1998.

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