Outcome of surgical treatment of pancreatic, peri-ampullary and ampullary cancer diagnosed in the south of The Netherlands: a cancer registry based study.

AIMS: To gain insight into the quality of pancreatic cancer surgery in 10 low-volume (median sized) hospitals, each serving 150,000-250,000 people, in the Comprehensive Cancer Centre South (CCCS) area and of referred patients to academic centres to determine the need for further regionalization. METHOD: The population-based Eindhoven Cancer Registry was used to select all patients in the CCCS area with pancreatic, peri-ampullary and ampullary cancer diagnosed between January 1, 1995 and April 30, 2000 (N = 1130). Of those, 124 patients (11%) underwent surgical resection (of which 40 were treated in university hospitals outside the region). RESULTS: For all pancreatic carcinoma resections, the 3-month survival rate was 82%, varying from 95% for referred patients to 76% for patients treated within the region (p = 0.014). One- and two-year survival rates showed no difference between both groups (p = 0.36 and p = 0.55, respectively). Surgically treated patients who were referred to university hospitals outside the CCCS area were younger, more often male, more often diagnosed with pTNM stage III, exhibited less comorbidity and had a higher socio-economic status than patients surgically treated within the region. CONCLUSION: Although the results are based on small numbers and patient selection probably influenced these outcomes, these data seem to support further hospital specialisation, to which the surgeons of the CCCS area have committed themselves.

[Compliance with the breast cancer guidelines in the region of the Comprehensive Cancer Centre South, 20031/'04]. / Naleving van mammacarcinoom-richtlijnen in de regio van het Integraal Kankercentrum Zuid, 2003/'04.

OBJECTIVE: To examine the level of compliance with the NABON-guidelines (i.e. breast cancer consensus recommendations) issued in 1999 with particular regard to the diagnostics and treatment of breast cancer in hospitals in the region covered by the Comprehensive Cancer Centre South (covering the Noord-Brabant and Noord-Limburg areas in the Netherlands). DESIGN: Retrospective, descriptive. METHOD: Using the Cancer Registry, the average number ofbreast cancer patients in 16 general hospital locations in the region covered by the Comprehensive Cancer Centre South was determined. Then, from I July 2003 to 30 June 2004, at each hospital location, all successive patients in whom carcinoma of the breast (invasive or in situ) had been diagnosed were included until one-third of the annual total was reached. Data from the medical-case notes of these patients were collected in order to examine to what extent the hospital locations had complied with the NABON-norms. RESULTS: A total of 581 breast cancer patients were included. In general the diagnostics and treatment complied with the consensus recommendations in the NABON-policy document. Improvements were mainly indicated in the area of logistics. One hospital met the guideline's recommendation that in 90% of cases, the pathology department should ensure that the results ofa histological needle-biopsy are available within 2 days of the biopsy being carried out. In 62% of patients, surgery was performed within 3 weeks of the necessity of an operation being confirmed, although the target norm was 90%. The interval between the last operation and the start of radiotherapy treatment was 44 instead of the proposed 28 days. Inter-hospital differences in diagnostics were seen mainly in the application of sentinel-node biopsy (34-95%). Furthermore, broad diversity was observed in the percentage of patients treated in the proposed space oftime between pathology result and initial surgery (3-87%) and between the last operation and start ofradiotherapy (0-46%) or chemotherapy (0-100%).

Expression of the follistatin/EGF-containing transmembrane protein M7365 (tomoregulin-1) during mouse development.

A novel transmembrane protein (designated X7365) containing two follistatin modules and an epidermal growth factor (EGF) domain has been described in the hypothalamic-pituitary axis of Xenopus laevis. We have now cloned the highly conserved mouse orthologue (M7365), and its mRNA was detected in many mesodermal and (neuro)ectodermal tissues in 8.5-day-old mouse embryos. During further development, M7365 mRNA expression became restricted to certain regions in the brain and to ganglia. In the adult mouse, the brain is the major site of M7365 expression.

Expression patterns of follistatin and two follistatin-related proteins during mouse development.

We compared the expression patterns of follistatin and two follistatin-related proteins (FRP and m7365) during early mouse development. m7365 is expressed continuously during preimplantation development, in contrast to FRP and follistatin. At early postimplantation stages, follistatin and 7365 are expressed from E6.0, while FRP is detected from E7.5 onwards. Although there is some overlap between the expression of these genes in the primitive streak and somites, their overall expression patterns are distinct.

Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors.

Activins are members of the transforming growth factor-beta family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-beta2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.

Developmental tumours, early differentiation and the transforming growth factor beta superfamily.

Embryonal carcinoma and embryonic stem cells have been very useful models for identifying some of the factors that regulate differentiation in early mammalian development. Here, we present a brief history of their original isolation and characterization and of their later introduction into the Hubrecht Laboratory. We illustrate in a review their contribution to our current understanding of the function of transforming growth factor beta and ligands binding to the receptors of a related factor, activin, in development with some of our own work.

Identification of a novel type II activin receptor, type IIA-N, induced during the neural differentiation of murine P19 embryonal carcinoma cells.

We have identified a novel type II activin receptor, called type IIA-N, the expression of which was induced during the neural differentiation of murine P19 embryonal carcinoma cells (P19 cells). P19 cells differentiate into several cell types dependent on the culture conditions. The induction of type IIA-N mRNA occurred predominantly in conjunction with neural differentiation. Sequence analysis of a cDNA clone for type IIA-N indicated that type IIA-N had a 24 bp insertion in the juxtamembrane region of the type IIA activin receptor suggesting that it is an alternative splicing product of the type IIA gene. Type IIA-N was also identified in human and Xenopus, and the amino acid sequences of three species were completely conserved. The expression of type IIA-N mRNA was specifically detected in neuroblastoma cells among several activin responsive cell lines. In vivo expression of type IIA-N mRNA was detected only in the neural tissues such as brain and spinal cord in adult mouse, by RT-PCR. Furthermore, its expression in developing Xenopus embryos was restricted to the neurula and later stages. These results suggest that the expression of type IIA-N is specific to neural cells and mediates neural differentiation-specific activin signaling.

Human growth-differentiation factor 3 (hGDF3): developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours.

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

Immunohistochemical localization and mRNA expression of activin, inhibin, follistatin, and activin receptor in bovine cumulus-oocyte complexes during in vitro maturation.

The aim of this study was to investigate whether bovine cumulus-oocyte complexes (COCs) synthesize activin A, inhibin, and follistatin and whether they contain activin receptor during in vitro maturation. Therefore, COCs obtained from small and medium-sized follicles were cultured in M-199 supplemented with 10% fetal calf serum (FCS) and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed for immunohistochemical staining to detect the expression of activin A, inhibin, follistatin, and activin receptor type II proteins. At 0 and 24 hr, COCs were removed and prepared for reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA of these proteins. It appeared that cumulus cells and oocytes express activin, follistatin, and activin receptor proteins as well as their mRNA. While expression of inhibin mRNA was found exclusively in cumulus cells, the inhibin protein was present in cumulus cells and oocytes. Immunohistochemical study both in cumulus cells and in oocytes often showed a moderate and strong staining intensity for activin and follistatin, respectively. Activin staining underwent little or no change during culture except at 24 hr of maturation, where about 60% of the oocytes showed no staining. Follistatin immunoreactivity remained strong in the majority of COCs. At the onset of culture, a spotlike inhibin staining was observed in the oocyte, which increased after 12 hr and was absent at the end of culture. Activin receptor immunoreactivity in cumulus cell membranes and oolemma increased during oocyte maturation to maximum values at the end of culture in most of the COCs. It is concluded that the consistent presence of activin and the increase in activin receptor in cumulus cells and oocytes during in vitro maturation indicate a paracrine and/or autocrine action for activin on bovine oocyte maturation. This action may be modulated by inhibin and/or follistatin.

DPC4 (SMAD4) mediates transforming growth factor-beta1 (TGF-beta1) induced growth inhibition and transcriptional response in breast tumour cells.

A family of structurally related proteins homologous to the Drosophila mothers against dpp (MAD) gene product have been implicated in signal transduction by members of the TGF-beta superfamily. One of these MAD related proteins (DPC4) has been cloned as a candidate tumour suppressor in pancreas carcinomas, suggesting a role for DPC4 in growth regulation by TGF-beta related proteins. The involvement of DPC4 in TGF-beta1 induced growth inhibition and transcriptional response is demonstrated here, by the introduction of DPC4 in the TGF-beta and activin insensitive breast tumour cell line MDA-MB-468, from which the DPC4 gene is deleted. Transfection of DPC4 in this cell line restores both growth inhibition and the induction of a TGF-beta sensitive reporter construct (3TPlux) by TGF-beta1. In contrast, a DPC4 splice variant lacking amino acid residues 223-301 and cloned from another TGF-beta and activin resistant breast tumour cell line (MDA-MB-231), does not restore the induction of the 3TPlux reporter by TGF-beta1. We also show that in this latter cell line activin resistance is partly due to the absence of a functional activin type IB receptor. These results indicate that DPC4 is part of the TGF-beta signalling cascade and mediates TGF-beta induced growth inhibition. Together with the deletion of DPC4 from pancreas carcinomas these results suggest a role for DPC4 as a tumour suppressor.

Transforming growth factor beta production during rat cytomegalovirus infection.

We analysed the production of transforming growth factor beta (TGF-beta) during a cytomegalovirus (CMV) infection in a rat model system. Splenocytes from immunocompetent rats infected with rat CMV (RCMV) released increased amounts of TGF-beta1. TGF-beta production was also evident in RCMV-infected radiation-immunosuppressed rats; their sera inhibited the interleukin 2-induced proliferation of T cells, which could be restored by anti-TGF-beta antibodies. In addition, TGF-beta production could be visualized immunohistologically in the lungs, spleen, liver and bone marrow of radiation-immunosuppressed infected rats. The virus directly induced this cytokine since TGF-beta was produced upon RCMV infection in vitro. The induction of TGF-beta production may contribute to immunosuppression during CMV infection.

Activin and basic fibroblast growth factor regulate neurogenesis of murine embryonal carcinoma cells.

Murine P19 embryonal carcinoma (EC) cells can be differentiated into various germ layer derivatives. The addition of retinoic acid (RA) to P19-EC cell aggregates results in a transient activation of receptor protein tyrosine phosphatase-alpha (RPTP alpha). Subsequent replating of these aggregates leads to neuronal differentiation. P19-EC cells expressing constitutively active RPTP alpha (P19-RPTP alpha) show extensive neuronal differentiation upon RA treatment in monolayer. P19-RPTP alpha cells thus provide a suitable in vitro model for studying neuronal differentiation. We used P19-RPTP alpha cells to study the effects of activin and basic fibroblast growth factor (bFGF) on neurogenesis. We show that P19-RPTP alpha cells express mRNA for types I and II activin receptors. RA addition causes an up-regulation of receptor type IIA expression. Complexes of type I and II receptors were detectable by cross-linking assays both before and after RA treatment. Receptor complexes were functional as determined by transient transfection assays with activin responsive reporter constructs. Undifferentiated as well as differentiated P19-RPTP alpha cells express also the FGF receptors (FGFRs) FGFR-1 and FGFR-2 but not FGFR-3 and FGFR-4. Their functionality was established by bFGF induced mitogen-activated protein kinase phosphorylation. Activin and bFGF appeared to exert differential actions on RA-induced neuronal differentiation. Although activin irreversibly changes the differentiation fate into nonneuronal directions, bFGF does not affect initial neurogenesis but regulates axonal outgrowth in a concentration-dependent way; low concentrations of bFGF enhance axonal outgrowth, whereas high concentrations inhibit this process. These results strengthen the notion that activin and bFGF are important regulators of neurogenesis in the mammalian embryo.

Regulation of the zebrafish goosecoid promoter by mesoderm inducing factors and Xwnt1.

Goosecoid is a homeobox gene that is expressed as an immediate early response to mesoderm induction by activin. We have investigated the induction of the zebrafish goosecoid promoter by the mesoderm inducing factors activin and basic fibroblast growth factor (bFGF) in dissociated zebrafish blastula cells, as well as by different wnts in intact embryos. Activin induces promoter activity, while bFGF shows a cooperative effect with activin. We have identified two enhancer elements that are functional in the induction of the goosecoid promoter. A distal element confers activin responsiveness to a heterologous promoter in the absence of de novo protein synthesis, whereas a proximal element responds only to a combination of activin and bFGF. Deletion experiments show that both elements are important for full induction by activin. Nuclear proteins that bind to these elements are expressed in blastula embryos, and competition experiments show that an octamer site in the activin responsive distal element is specifically bound, suggesting a role for an octamer binding factor in the regulation of goosecoid expression by activin. Experiments in intact embryos reveal that the proximal element contains sequences that respond to Xwnt1, but not to Xwnt5c. Furthermore, we show that the distal element is active in a confined dorsal domain in embryos and responds to overexpression of activin in vivo, as well as to dorsalization by lithium. The distal element is to our knowledge the first enhancer element identified that mediates the induction of a mesodermal gene by activin.

Active complex formation of type I and type II activin and TGF beta receptors in vivo as studied by overexpression in zebrafish embryos.

We have investigated the involvement of activin receptors and TGF beta type I receptor in zebrafish development. Overexpression of either full-length or a truncated form of mouse ActR-IIA interferes with the development. Different splice variants of mouse ActR-IIB have distinct effects; ActR-IIB4 induces abnormal embryos, whereas ActR-IIB2 does not. Activin and TGF beta type I receptors can induce axis duplications. Co-expression of ActR-IA or ActR-IB with the type II activin receptors results in a synergistic increase of the frequency of axis duplication. Moreover, ActR-IIB2 is synergistic with ActR-IA and ActR-IB, demonstrating that ActR-IIB2 can interact with the zebrafish ligand. Overexpression of TGF beta R-I with ActR-IIA or ActR IIB4 results in a synergistic increase in frequency of abnormal embryos, whereas in combination with ActR-IIB2 no such increase occurs.

Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors.

Follistatin is an activin-binding protein, which inhibits activin bioactivity in several biological systems. In the present study it is demonstrated that preincubation of iodinated activin A with follistatin, purified from porcine follicular fluid, completely abolished the binding of activin to activin type IIA, IIB2 and IIB4 receptors, and consequently to activin type IB receptor, transiently transfected in COS cells. Binding of activin A to membrane proteins on the activin-responsive P19 embryonal carcinoma cells was also prevented by this follistatin preparation. The same results were obtained with a carboxy-terminally truncated form of follistatin (FS-288), which is only present in minor amounts in the purified follistatin preparation. Since FS-288 has a high affinity for heparan sulfate proteoglycans on the cell surface, we tested whether membrane-bound FS-288 presents activin A to the different activin receptors, thereby facilitating activin binding. FS-288 did bind to the cell surface of transfected COS cells, but inhibited the binding of activin A to its receptors IIA, IIB2 and IIB4. Furthermore, after addition of FS-288 to K562 erythroleukemia cells, the total binding of activin via cell surface-bound FS-288 was increased, whereas the binding of activin A to activin type II and type I receptors present on these cells was inhibited. These findings reveal that different forms of follistatin can neutralize activin bioactivity by interference with binding of activin to all known activin type II receptors, rather than that they inhibit the binding of the type I receptor to the activin/activin type II receptor complex. In addition, our studies indicate that cell surface-associated follistatin cannot present ligand to signalling receptors.

Resistance to transforming growth factor beta and activin due to reduced receptor expression in human breast tumor cell lines.

Loss of sensitivity to growth inhibition by transforming growth factor (TGF)-beta is a phenomenon often observed in human epithelial tumor cells and is linked to malignant progression. We tested a panel of estrogen receptor (ER)-positive and -negative breast cell lines for their sensitivity to TGF-beta and a related member of the TGF-beta superfamily, activin. Both TGF-beta-sensitive (MCF7, Hs578T, and BT20) and -resistant (two T47D variants, ZR75-1, MDA-MB231, and MDA-MB468) cell lines were found, with no strict correlation between ER content and sensitivity to TGF-beta. In contrast, all four ER-positive cell lines were inhibited by activin A, whereas the ER-negative lines were not. To examine whether resistance to TGF-beta and activin resulted from the absence of the corresponding receptors, mRNA expression of the types I and II receptors was studied. TGF-beta receptor II was not expressed in the two T47D variants and was low in ZR75-1 cells. Upon stable transfection of the TGF-beta receptor II in one of the T47D variants, sensitivity to TGF-beta 1 and TGF-beta 2 was restored with respect to inhibition of anchorage-dependent and -independent proliferation, indicating that other signal transduction components are functionally intact. Sensitivity to TGF-beta in the transfectants was dependent on the expression level of the newly introduced receptor. Resistance to activin in the ER-negative cell lines could be explained in BT20 and Hs578T cells, but not in MDA-MB231 and MDA-MB468, by low activin receptor expression. These results show that resistance to TGF-beta and activin is often, but not always, due to reduced expression of the signaling receptor in breast cancer cells. The activin resistance of ER-negative breast tumor cells may be involved in their increased malignancy compared with ER-positive cells.

Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors.

The role of TGF-beta production in growth inhibition of breast-tumor cells by progestins.

We have studied the influence of synthetic progestins on the estrogen-induced proliferation and type-beta transforming-growth-factor (TGF-beta) production of 3 breast-tumor cell lines. In long-term growth experiments, progestins inhibited proliferation of T47D cells, while a specific T47D variant and MCF7 cells were not affected, despite the presence of functional progesterone receptors. The effect of progestins was biphasic, since an initial stimulation of proliferation was followed by a prolonged inhibition. This response suggests the involvement of a progestin-induced negative growth regulator. We show here that TGF-beta s do not fulfill this role since (i) the progestin-induced T47D cells are not sensitive to TGF-beta 1, -beta 2 or -beta 3, (ii) secretion of TGF-beta s is decreased by progestins in all 3 cell lines, and (iii) TGF-beta neutralizing antibodies do not reverse progestin-induced growth inhibition. Furthermore, evidence was obtained that medium conditioned by T47D cells does not contain any other growth inhibitor to which this cell line responds in a negative autocrine manner. In contrast, MCF7 cells are growth-inhibited by all 3 TGF-beta isoforms, but are not growth-inhibited by progestins, suggesting that there is no correlation between growth inhibition by progestins and responsiveness to and production of TGF-beta in vitro. Although TGF-beta is a strong growth inhibitor of normal mammary tissue, recent evidence suggests that, in malignant tissue, enhanced TGF-beta secretion correlates with increased malignancy. Therefore, a progestin-induced decrease in TGF-beta production, as observed here, may lead to enhanced proliferation of normal but not malignant mammary epithelium.

Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins.

Using in situ hybridization we have studied the localization of the messenger RNAs encoding the inhibin/activin subunits (alpha, beta A, beta B), the activin-binding protein follistatin and activin receptors (IIA, IIB) in mouse embryos during postimplantation development. From 6.5- to 9.5-days post coitum (p.c.) activin beta A and beta B subunit expression was restricted to the decidua, while activin receptor type IIB messages were exclusively detected in the embryo. Expression of activin receptor type IIA was apparent in the embryo as early as 9.5 days p.c. In contrast, follistatin transcripts were present in both the decidua and the embryo at the early postimplantation stages. In particular, the primitive streak region, specific rhombomeres in the developing hindbrain, somites, paraxial mesoderm and parietal endoderm cells attached to the Reichert's membrane showed strong expression of follistatin. In 10.5- and 12.5-day embryos expression of the beta A subunit message was abundant in mesenchymal tissue, in particular in the developing face, the body wall, the heart, precartilage condensations in the limb and in the mesenchyme of structures that show both epithelial and mesenchymal components, including tissues of the embryonic digestive, respiratory and genital tracts. The distribution of beta B transcripts was quite different from that observed for beta A. beta B is strongly expressed in selected regions of the brain, in particular the fore- and hindbrain, and in the spinal cord. Specific hybridization signals were also present in the epithelium of the stomach and oesophagus. Common sites of beta A and beta B expression are blood vessels, intervertebral disc anlagen, mesenchymal condensations in the flank region and the gonad primordium. The latter organ is the only site in the embryo where the alpha subunit is expressed, and thus where inhibit activity may be present. During the period of organogenesis the sites of expression of activin receptors type IIA and IIB messenger RNA (mRNA) generally coincide with or are adjacent to the sites of beta subunit expression. Differences in the expression patterns of the receptor RNAs are the whisker follicles, where type IIA is expressed, and the metanephros and the forebrain where type IIB transcripts are present. Taken together, the present data suggest that follistatin, but not one of the known activin forms (A,B,AB) is involved in early postimplantation development.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of activin subunits in early murine development determined by subunit-specific antibodies.

Activins are thought to be involved in early differentiation processes during amphibian and avian development. Little is known, however, about the role of activins in early developmental stages of higher vertebrates. In order to study activin protein expression in early murine development we have prepared polyclonal antibodies against synthetic peptides corresponding to C-terminal sequences of the murine beta A and beta B activin subunits. Both antibodies recognized specifically activin A and activin B in different immunological assays including ELISA and immunoblotting. Using these antibodies in immunofluorescence experiments a good correlation between mRNA and protein expression of the beta subunits was observed in different in vitro model systems for early murine development. In addition, beta A and beta B subunit protein localization appeared to be different in 3.5- and 4.5-day mouse blastocysts. The results presented here are consistent with a role for different activin forms in the regulation of distinct processes in early murine development.