Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.     

Immunolocalisation of aspartic proteinases in the developing human stomach

The distribution and time of appearance in the developing human stomach of the 4 aspartic proteinases, pepsinogen, progastricsin, slow-moving protease and cathepsin D, all present in gastric carcinoma, has been determined by the peroxidase-antiperoxidase method on formalin fixed paraffin embedded sections of fetal stomach. Slow-moving protease appears to be the dominant enzyme from 12 weeks gestation onward, although progastricsin is also present at this time. Pepsinogen and cathepsin D do not appear until 17-18 weeks.     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

Further studies on rat cathepsin E: subcellular localization and existence of the active subunit form

The subcellular localization of rat neutrophil cathepsin E was examined by a modification of the method of N. Borregaard et al. [(1983) J. Cell Biol. 97, 52-61]. When the postnuclear cavitate of rat neutrophils was subjected to density centrifugation on discontinuous Percoll gradients, three particulate bands, P1 (lowest; azurophil granule rich), P2 (middle; specific granule rich), and P3 (highest; plasma membrane rich), were segregated. A combined application of immunochemical and electrophoretic methods revealed a striking difference in subcellular localization between cathepsin E and cathepsin D: Cathepsin E was associated with P3 and soluble fractions, and cathepsin D was chiefly associated with P1 and P2 fractions. The results thus indicate that cathepsin E is a nonlysosomal acid proteinase in rat neutrophils. It was found that cathepsin E existed in two enzymatically active molecular forms, referred to as CE-I and CE-II, in rat neutrophil extracts. To examine the relationships between the two forms, cathepsin E was purified to homogeneity from rat gastric mucosae. The purified enzyme exhibited a single protein band of 43 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, but electrophoresis without SDS, followed by visualization of activity in the gel, revealed two activity bands corresponding to CE-II and CE-I in neutrophil extracts. Pretreatment of the enzyme with beta-mercaptoethanol or dithiothreitol resulted in an increase in CE-I activity with a concomitant decrease in CE-II activity on gels. Upon gel filtration, the molecular weights of CE-II and CE-I were estimated to be 98,000 and 51,000, respectively, strongly suggesting that they are the dimeric and monomeric forms of the cathepsin E subunit.     

Synthesis and intracellular distribution of cathepsins E and D in differentiating murine Friend erythroleukemia cells.

The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship.     

A comparative study of two kinds of cathepsin D-type proteinases from rat gastric mucosa

The localization of cathepsin D-like acid proteinase in the rat stomach and other tissues was studied, and its biochemical properties were compared with those of rat gastric cathepsin D (EC 3.4.23.5). Cathepsin D-like acid proteinase existed overwhelmingly in the mucosal layer and was hardly detected in the gastric juice. Its subcellular distribution profile was very similar to that of acid phosphatase, but not to that of pepsinogen. This proteinase-like enzyme activity was also found in rat splenic extract. These results strongly suggest that the proteinase is a lysosomal enzyme. In addition, cathepsin D-like acid proteinase demonstrated an in vitro transition of molecular species during storage at -30 degrees C. Although this molecular change was distinctive in ion-exchange column chromatography and susceptibility to some enzyme inhibitors, it was not accompanied by a significant decrease in molecular weight. To compare cathepsin D-like acid proteinase with ordinary cathepsin D, gastric cathepsin D was newly purified to apparent homogeneity in polyacrylamide gel electrophoresis. Its biochemical properties demonstrate that this is a true cathepsin D in rat gastric mucosa. Moreover, this cathepsin D activity was not abolished by treatment with antiserum specific to cathepsin D-like acid proteinase or pepsinogen. From these results, we can conclude that the proteinase is a lysosomal acid proteinase different from newly purified gastric cathepsin D.     

Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung

High affinity (Kd=0.2 nM), low capacity (48 fmoles per mg protein), stereospecific binding sites, with properties characteristic of the β₁-subtype of β-adrenergic receptors, have been detected in fetal rabbit lung membranes as early as the 22nd day of gestation. The concentration of the receptor did not change significantly between the 22nd and 26th day of gestation, but increased 3-fold between the 26th and 29th day, reaching a level of 198 fmoles per mg protein. A further increase (from 198 to 315 fmoles per mg protein) in receptor concentration was observed in adult female rabbits. The increase in the levels of pulmonary β-adrenergic receptors between the 26th and 29th day of gestation is temporally related to the increase in surfactant release into the alveolar spaces of the fetal lung. Thus β-adrenergic agonists may act directly on the fetal lung to regulate surfactant secretion.     

The expression and function of cathepsin E in dendritic cells

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.     

Immunochemical similarity between a gastric mucosa non-pepsin acid proteinase and neutrophil cathepsin E of the rat

Antiserum against a rat gastric mucosa non-pepsin acid proteinase precipitates rat neutrophil cathepsin E, with a precipitation curve essentially similar to that of the gastric enzyme. Taken together that the antiserum precipitates a cathepsin E-like acid proteinase from rat spleen (Muto, N., Yamamoto, M. and Tani, S. (1987) J. Biochem. (Tokyo) in press), the data indicate that the non-cathepsin D acid proteinases in rat neutrophils, gastric mucosa and spleen are immunochemically closely related. In contrast with the earlier data, cathepsin E from rabbit neutrophils exhibited a maximal activity at around pH 3.0-3.2 and preferred hemoglobin to albumin as substrate, which supports that the non-cathepsin D acid proteinases in the rat tissues are relevantly classified as cathepsin E.     

Increase in 15-hydroxyprostaglandin dehydrogenase activity in the ovine placentome at parturition and effect of oestrogen

Type 1 NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is the key enzyme for metabolism of active primary prostaglandins to inactive forms in gestational tissues. The present study examined the activity and immunolocalization of PGDH in the ovine placenta, fetal membranes and uterus over the latter half of pregnancy, and its potential regulation by oestradiol. Placenta, fetal membranes and myometrium were collected from sheep with known single insemination dates on days 70, 100 and 135 of gestation and in active labour demonstrated by electromyographic activity. In addition, chronically catheterized fetuses were infused with oestradiol (100 microgram kg(-1) per 24 h) (n = 5) or saline vehicle into the fetus from day 120 to day 125. PGDH activity measured in placental extracts remained constant from day 70 to day 135 of gestation, and then significantly (P < 0.05) increased by 300% in active labour. Immunoreactive PGDH was localized in the placentome at all stages and was present predominantly in the fetal component of the placentome in uninucleate, but not in binucleate, trophoblast cells. Similarly, in the fetal membranes PGDH immuno-reactivity was present in the uninucleate trophoblast but not in the binucleate cells of the chorion. PGDH immunostaining was also present in the endometrial luminal epithelium, in the smooth muscle of the myometrium, and the glandular epithelium of the cervix. Infusion of oestradiol into the fetal circulation from day 120 to day 125 of gestation had no effect on placental PGDH activity. Immunohistochemistry was used to localize oestrogen receptor alpha in intrauterine tissues to investigate further the failure of oestradiol to increase PGDH activity. Immunoreactive oestrogen receptor alpha was not present in the fetal component of the placenta, although it was expressed in adjacent maternal-derived cells. It is concluded that (1) PGDH activity increases in late gestation; (2) PGDH is expressed in uninucleate trophoblast cells in the ovine placenta and fetal membranes, and also in the maternal endometrial epithelium and stroma, myometrium and cervix; (3) oestrogen receptor alpha is not expressed in fetal cells in the placenta or fetal membranes; and (4) the increase in PGDH activity is not regulated by oestradiol administered to the fetus.     

Metabolomic evaluation of di-n-butyl phthalate-induced teratogenesis in mice

Di-n-butyl phthalate (DBP) has been linked to the neural, reproductive and developmental toxicity. We present here a metabolomic study that characterized the metabolic variations associated with the DBP-induced teratogenesis in maternal and fetal mice. DBP at 50 and 300?mg/kg were administrated to pregnant C57 mice, via gastric intubation on gestation day 7?C9, respectively. Maternal mice were euthanized on gestation day 16 and examined for fetal development and malformations. Metabolomic study of maternal serum, placenta and fetal brain tissues was performed using gas chromatography time-of-flight mass spectrometry combined with multivariate data analysis (MVDA). The results showed that a 50?mg/kg dose of DBP had no significant effect on fetal development and a 300?mg/kg dose caused embryo resorption and fetal malformations (primarily eye abnormalities and encephalocele). MVDA indicated that DBP at two doses gave rise to disruption of maternal and fetal metabolic profiles characterized by significantly altered tricarboxylic acid cycle, amino acid, purine and lipid metabolism.     

Emerging roles of cathepsin E in host defense mechanisms

Cathepsin E is an intracellular aspartic proteinase of the pepsin superfamily, which is predominantly expressed in certain cell types, including the immune system cells and rapidly regenerating gastric mucosal and epidermal keratinocytes. The intracellular localization of this protein varies with different cell types. The endosomal localization is primarily found in antigen-presenting cells and gastric cells. The membrane association is observed with certain cell types such as erythrocytes, osteoclasts, gastric parietal cells and renal proximal tubule cells. This enzyme is also found in the endoplasmic reticulum, Golgi complex and cytosolic compartments in various cell types. In addition to its intracellular localization, cathepsin E occurs in the culture medium of activated phagocytes and cancer cells as the catalytically active enzyme. Its strategic expression and localization thus suggests the association of this enzyme with specific biological functions of the individual cell types. Recent genetic and pharmacological studies have particularly suggested that cathepsin E plays an important role in host defense against cancer cells and invading microorganisms. This review focuses emerging roles of cathepsin E in immune system cells and skin keratinocytes, and in host defense against cancer cells. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Properties and intracellular distribution of a cathepsin D-like proteinase active at the acid region of Musca domestica midgut

Musca domestica larval midgut display in cells and luminal contents a proteolytic activity with a pH optimum of 3.0–3.5. This activity is abolished by pepstatin and is insensitive to soybean trypsin inhibitor and to sulfhydryl proteinase inhibitors. The acid proteinase occurs in multiple forms with M_r values in the range 40,000–80,000 and with pI values of about 5.5. The proteinase inactivates at 60°C according to apparent first-order kinetics and Lineweaver-Burk plots of its activity against albumin concentration are rectilinear, suggesting that the multiple forms have similar properties. The proteinase reacts slowly with diazoacetylnorleucine plus CuSO₄, is stable in alkaline media, is inhibited by dithiothreitol, hydrolyses hemoglobin better than albumin and is virtually not active upon synthetic substrates for pepsin. These properties are similar to those of cathepsin D. The specific activity of the acid proteinase determined by titration with pepstatin is 680 units/mg of proteinase and the K_D of the pepstatin-proteinase complex is 1.5 nM at 30°C. The acid proteinase occurs mainly in midgut subcellular fractions characterized by a high specific activity of molybdate-inhibited acid phosphatase and a large number of secretory-like vesicles. It is proposed that the M. domestica midgut acid proteinase is a cathepsin D-like proteinase evolved to function in luminal contents. The lack of ATP activation of the midgut enzyme supports this hypothesis, since ATP is thought to regulate cathepsin D-proteolysis inside lysosomes.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.     

Differential regulation of the nature and functions of dendritic cells and macrophages by cathepsin E

The aspartic proteinase cathepsin E is localized mainly in the endosomal structures of APCs and has been implicated in a variety of immune responses, however, the precise roles of cathepsin E in these cells remain speculative. In this study, we report the effect of disrupting the gene encoding cathepsin E on the nature and functions of dendritic cells (DCs) and macrophages derived from mouse bone marrow precursors, as well as mouse peritoneal macrophages. Whereas cathepsin E deficiency induced the accumulation of the lysosome-associated membrane protein (LAMP)-1 and LAMP-2 and elevated the lysosomal pH in macrophages, it did not have these effects on DCs. Although cathepsin E deficiency also caused a marked decrease in degradation of phagocytosed OVA and chemotactic responses to MCP-1 and fMLP by macrophages, these abilities were little affected in DCs by the absence of cathepsin E. Interestingly, cathepsin E deficiency markedly decreased the ability of macrophages to present intact OVA, as well as an OVA-derived antigenic peptide (266-281), to cognate T cells, while that of DCs was inversely enhanced by the absence of this protein. This paradox was resolved, in part, by the enhanced phagocytic activity and the increased expression of the costimulatory molecules CD86, CD80, and CD40, which amplify the response of T cells, in cathepsin E-deficient DCs compared with the wild-type cells. These results indicate that cathepsin E differentially regulates the nature and function of DCs and macrophages.     

Expression of nitric oxide synthase isoforms is reduced in late-gestation ovine fetal brainstem

Fetal baroreflex responsiveness increases in late gestation. An important modulator of baroreflex activity is the generation of nitric oxide in the brainstem nuclei that integrate afferent and efferent reflex activity. The present study was designed to test the hypothesis that nitric oxide synthase (NOS) isoforms are expressed in the fetal brainstem and that the expression of one or more of these enzymes is reduced in late gestation. Brainstem tissue was rapidly collected from fetal sheep of known gestational ages (80, 100, 120, 130, 145 days gestation and 1 day and 1 wk postnatal). Neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) mRNA was measured using real-time PCR methodology specific for ovine NOS isoforms. The three enzymes were measured at the protein level using Western blot methodology. In tissue prepared for histology separately, the cellular pattern of immunostaining was identified in medullae from late-gestation fetal sheep. Fetal brainstem contained mRNA and protein of all three NOS isoforms, with nNOS the most abundant, followed by iNOS and eNOS, respectively. nNOS and iNOS mRNA abundances were highest at 80 days' gestation, with statistically significant decreases in abundance in more mature fetuses and postnatal animals. nNOS and eNOS protein abundance also decreased as a function of developmental age. nNOS and eNOS were expressed in neurons, iNOS was expressed in glia, and eNOS was expressed in vascular endothelial cells. We conclude that all three isoforms of NOS are constitutively expressed within the fetal brainstem, and the expression of all three forms is reduced with advancing gestation. We speculate that the reduced expression of NOS in this brain region plays a role in the increased fetal baroreflex activity in late gestation.     

Variability in the developmental toxicity of bropirimine with the day of administration

The aim of this study was to determine the mechanism by which bropirimine exerts its developmental toxicity. This drug is an immunomodulator and interferon inducer with antiviral and antitumor activities in experimental models. Timed-pregnant Upj:TUC(SD)spf (Sprague-Dawley) rats were given a single oral (gastric intubation) dose of bropirimine at 200 or 400 mg/kg (doses as high as 100 mg/kg/day have been employed in human cancer trials) on days 5, 6, 7, 8, 9, 10, 11, or 12 of gestation and in a second experiment on day 12, 13, 14, 15, 16, 17, 18, or 19 of gestation. The dams were killed 24 hours after dosing and their uterine contents examined. In a third experiment, bropirimine (400 mg/kg) was administered on day 4 of gestation and the uteri of different groups were examined on day 8, 9, 10, 11, or 12 of gestation. Serum progesterone levels were measured at sacrifice. In the first two experiments a battery of hematologic/clinical chemistry assays also were performed. In all three experiments, bropirimine-related maternal toxicity was observed; such toxicity was characterized by significant decreases in weight gain, relative to the concurrent vehicle controls, as well as significant differences in several blood parameters including platelets, white blood cells, alanine aminotransferase, and aspartate transaminase. In the first experiment, bropirimine treatment on day 11, but not day 12, resulted in significant decreases in the mean number of live embryos per litter. In the second experiment, significant decreases in the number of live fetuses per litter occurred 24 hours after dosing on day 18 (200 and 400 mg/kg groups) or day 19 (400 mg/kg group). Decreases in serum progesterone appeared to correlate well with the embryolethal effects seen after treatment between days 6 and 11 of gestation, but not with the fetal lethality seen when treatment was given on day 17 or 18. The decreases in serum progesterone levels found most likely were the result of a luteolytic effect, although it is unknown if bropirimine has a direct or indirect effect on the corpora lutea. In the third experiment, bropirimine treatment on day 4 of gestation resulted in only slight preimplantational losses, but significant decreases were found in mean number of live embryos per litter after day 9. Uterine decidual necrosis has been observed in the first experiment where bropirimine was given on day 11; however, treatment on day 4 resulted in an apparent decrease in decidual development but not necrosis.