Gilbert A. Schultz

Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues

We have isolated cDNA clones corresponding to a new member of the murine tissue inhibitor of metalloproteinase (TIMP) family, designated Timp‐4. The nucleotide sequence predicts a protein of 22 609 Da that contains the characteristic 12 cysteine TIMP signature. TIMP‐4 is more closely related to TIMP‐2 and TIMP‐3 than to TIMP‐1 (48%, 45% and 38% identity, respectively). Analysis of Timp‐4 mRNA expression in adult mouse tissues indicated a 1.2 kb transcript in brain, heart, ovary and skeletal muscle. This pattern of expression distinguishes Timp‐4 from other Timps, suggesting that the TIMP‐4 protein may be an important tissue‐specific regulator of extracellular matrix remodelling.

Expression analysis of the entire MMP and TIMP gene families during mouse tissue development

Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane‐bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis☆

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.

Changes in the quantity of histone and actin messenger RNA during the development of preimplantation mouse embryos

Actin and histone H3 mRNA levels in mouse eggs and early embryos have been measured by use of recombinant DNA probes having sequence homology to those mRNA species. Total nucleic acid was extracted from pools of unfertilized eggs, two-cell embryos, eight-cell embryos, and blastocysts. The nucleic acids were resolved electrophoretically, bound to diazotized paper following Northern transfer, and hybridized with 32P-labeled histone or actin DNA probes. Our findings demonstrate that there is a maternal store of histone and actin mRNA in the unfertilized egg but that this mRNA pool is reduced roughly 10-fold on an embryo basis by the mid-two-cell stage. Following this reduction of maternal mRNA, histone and actin mRNA accumulation from the eight-cell cleavage stage to the blastocyst increases proportionally to cell number and appears to be controlled by zygote genome transcription.

Gene expression in pre-implantation mammalian embryos

The pre-implantation mammalian embryo is initially under the control of maternal informational macromolecules that are accumulated during oogenesis. Subsequently, the genetic program of development becomes dependent upon new transcription derived from activation of the embryonic genome. Several embryonic transcripts including those that encode growth factors, cell junction components and plasma membrane ion transporters are required for normal progression of the embryo to the blastocyst stage. The pattern of genes expressed and the overall program of development is subject to the influences of genomic imprinting as well as external influences encountered by the embryo within the maternal reproductive tract.

Tissue inhibitor of metalloproteinases-3 is the major metalloproteinase inhibitor in the decidualizing murine uterus

Embryo implantation in the mouse is a highly orchestrated process, a key aspect of which is the invasion of trophoblast cells of the blastocyst into the maternal uterine endometrium. Invasion is facilitated via proteinases expressed by trophoblast cells and balanced by expression of inhibitors of proteinases in the maternal decidua. The predominant proteinase expressed by trophectodermal derivatives of the implanting mouse embryo is matrix metalloproteinase‐9 (MMP‐9; gelatinase B). Using in situ hybridization, transcripts for MMP‐9 were detected in trophoblast cells of the embryo from the earliest stage of decidual formation (day 6.0) examined. MMP‐9 transcripts were localized to trophoblast giant cells at the periphery of the embryo at the egg cylinder stage (day 7.0). By the neural‐fold stage (day 8.5), expression was restricted to giant cells adjacent to the maternal side of the developing placenta, and by day 9.5 few MMP‐9‐positive cells remained. The major tissue inhibitor of metalloproteinases (TIMP) produced during this period was TIMP‐3. Transcripts encoding TIMP‐3 were detected from day 6.0–7.0 in the maternal decidua immediately adjacent to embryonic cells expressing MMP‐9. The intensity of TIMP‐3 expression in later‐stage embryos declined in parallel with MMP‐9 expression. Maternal TIMP‐3 expression also occurred in the absence of embryonic MMP‐9 expression in decidual reactions induced by parthenogenetic embryos (where MMP‐9 positive cells were not detected) or in oil‐induced deciduomas. These results support the hypothesis that MMP‐9 is an important mediator of cellular invasiveness during embryo implantation, and that TIMP‐3 serves as a regulator within the uterus to restrict invasion to the site of implantation.

The POU homeodomain protein OCT3 as a potential transcriptional activator for fibroblast growth factor-4 (FGF-4) in human breast cancer cells.

The POU (representing a homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86) homeodomain protein OCT3/Oct-3 (where OCT stands for octamer-binding protein) is an embryonic transcription factor expressed in oocytes, embryonic stem and embryonic carcinoma cells. We have demonstrated previously that human breast cancer cells regain the ability to express OCT3 mRNA [Jin, Branch, Zhang, Qi, Youngson and Goss (1999) Int. J. Cancer 81, 104-112]. Antibodies against human OCT3 were not available when this study was conducted. By using a human OCT3-glutathione S-transferase fusion protein to affinity purify a polyclonal antibody against the mouse Oct-3, we obtained an antibody that enabled us to detect OCT3 in human breast cancer cells by Western-blot analysis. Thus we have now confirmed that OCT3 is expressed in human breast cancer cells but not in normal human breasts and in three other organs. When breast cancer cell lines were treated with all- trans -retinoic acid, OCT3 expression was repressed, associated with decreased cell proliferation. Although another POU protein Brn-3 has been shown to be a repressor for BRCA1 (breast-cancer susceptibility gene 1), OCT3 does not repress human or mouse BRCA1/Brca-1 promoters. However, OCT3 is capable of activating a fusion promoter containing the fibroblast growth factor-4 (FGF-4) enhancer element. In addition, we documented for the first time that human breast cancer cells express FGF-4 protein, and its expression could be inhibited by all- trans -retinoic acid. Furthermore, overexpressing OCT3 stimulated endogenous FGF-4 expression in MCF7 breast cancer cell line. These observations indicate that OCT3 protein is selectively expressed in human breast cancer cells, and its expression may be implicated in mammary gland tumorigenesis via up-regulating FGF-4 expression.

Expression of proteinases and proteinase inhibitors during embryo‐uterine contact in the pig

Implantation in pigs is noninvasive and characterized by interdigitation of embryonic and endometrial epithelial cell processes. However, when pig embryos are transferred to ectopic sites, trophoblast becomes invasive. The objective of this study was to evaluate expression of proteinases and proteinase inhibitors in pig embryos and uteri at the time of endometrial attachment. RNA was extracted from Day 15.75 pig embryos and uteri and reverse transcribed, and cDNA was amplified by polymerase chain reactions using primers specific for urokinase-type plasminogen activator (uPA), matrix metalloproteinases-2 and -9 (MMP-2 and -9), and tissue inhibitors of MMP-1, -2, and -3 (TIMP-1, -2, and -3). Localization of transcripts for the genes of interest in embryos and uteri was performed using in situ hybridization with antisense riboprobes. Day 15.75 pig embryos and uteri expressed transcripts for uPA, MMP-2 and -9, and TIMP-1, -2, and -3. In situ hybridization revealed weak expression of uPA in the trophectoderm and moderate expression in the adjacent extraembryonic endoderm. TIMP-1 transcripts were abundant in extraembryonic endoderm and scattered throughout the trophectoderm. TIMP-2 appeared to be expressed in all cells of the embryo. TIMP-3 expression was observed in the trophectoderm and, to a lesser extent, in the extraembryonic endoderm. Specific localization of MMP-2 and -9 transcripts above background was not observed by in situ hybridization in either embryos or uterus. Uterine expression of uPA and TIMP-1, -2 and -3 was localized to the endometrial stroma. Transcripts of these genes were not observed in either the luminal or glandular endometrial epithelium. These results suggest that pig embryos and uteri express a wide array of proteinases and proteinase inhibitors during the period of uterine association. The abundant expression of proteinases and proteinase inhibitors during the period of uterine association. The abundant expression of TIMP in pig embryos may partially explain the absence of invasive implantation in this species in contrast to implantation typified by rodents and primates.

Changes in the relative abundance of various housekeeping gene transcripts in in vitro-produced early bovine embryos.

The relative abundances of transcripts of different origins and housekeeping functions were measured by Northern blot analysis of RNA samples derived from in vitro‐matured oocytes and in vitro‐produced bovine embryos at selected stages of early development. The gene products studied included: two mitochondrial transcripts, 12S rRNA and cytochrome b mRNA; two RNAs involved in the processing of other RNAs, U2 and U3 snRNA; and two nuclear‐derived transcripts, β‐actin mRNA and histone H3 mRNA. Overall, the RNA levels for the various genes studied remained constant or decreased slightly from the mature oocyte to the 6‐ to 8‐cell or morula stage and were greatly increased in blastocysts. Differences were observed in the degree to which the RNA levels increased and in the timing of the increase. For 12S rRNA, a major increase was not observed until the blastocyst stage where levels increased 7.1 times the amount detected in morulae. Cytochrome b mRNA levels started to increase at the 6‐ to 8‐cell stage and reached levels in blastocysts that were 20 times more than the cytochrome b mRNA level in 2‐ to 4‐cell embryos. U2 snRNA levels did not increase until the blastocyst stage where levels were 6.4 times the amount found in morulae. U3 snRNA and β‐actin mRNA levels started to increase at the morula stage and blastocysts contained 118 and 110 times more U3 snRNA and β‐actin mRNA, respectively, than 6‐ to 8‐cell embryos. However, blastocysts contained only two times the amount of histone H3 mRNA present in 6‐ to 8‐cell embryos. Mol. Reprod. Dev. 47:413–420, 1997.

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.