Regulation of uterine 5 alpha-reductase type 1 in mice.

A null mutation in the murine gene encoding steroid 5 alpha-reductase type 1 (5 alpha R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5 alpha R1 increase significantly at term in normal pregnant animals, indicates that 5 alpha R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5 alpha R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17 beta-estradiol (E(2)), progesterone (P(4) ), or E(2)+P(4) for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5 alpha R1 specific activity, and Northern blot analysis of 5 alpha R1 mRNA expression. The 5 alpha R1 enzyme activity was significantly increased in animals treated with E(2)+P(4). However, activity was much less in uterine tissues from E(2)+P(4)-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5 alpha R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5 alpha R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5 alpha R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5 alpha R1 is regulated by E(2)+P(4) in uterine tissues. Whereas E(2) alone is insufficient to induce enzyme activity, E(2) may be required to increase P(4) receptors and, thereby, mediate the effects of P(4) on 5 alpha R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5 alpha R1 is regulated by both hormonal and fetal-derived signaling pathways.     

Structural analysis of the murine IgG3 constant region gene.

We have determined the complete sequence of the gamma 3 heavy chain constant (C gamma 3) region gene of the BALB/c mouse including the 5''-flanking region up to the switch site and the 3''-flanking region past the end of the membrane exons. The C gamma 3 coding region, typical of other IgGs, is divided into six exons corresponding to the protein domains (C gamma (3)1, hinge, C gamma (3)2, and C gamma (3)3) and to the membrane carboxyl terminus (M1 and M2). The predicted amino acid sequence of the gamma 3 chain has three potential N-linked carbohydrate addition sites (including one in the membrane spacer segment), as compared with a single occurrence in the other mouse IgGs. Between the switch recombination region and the body of the C gamma 3 gene, there is a remarkable homology with a sequence between C mu and C delta which provides a rationale for an alternative, T cell-independent class-switch mechanism. We have used a computer to analyze the secondary structure of the gamma 3 mRNA precursor for the membrane form. We predict that this RNA precursor (approximately 12 000 bp) folds into four leaf-like domains which correspond to the variable region, the large IVS, the body of the constant region, and the membrane exons. This organization may have a role to play in the function of the mRNA precursor.     

Fetal Fibronectin Signaling Induces Matrix Metalloproteases and Cyclooxygenase-2 (COX-2) in Amnion Cells and Preterm Birth in Mice

Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E₂ synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes.     

Electrokinetic Properties of the Sarcoplasmic Reticulum Membrane Obtained from Reconstitution Studies

Electrophoretic mobility data of SR vesicles reconstituted with uncharged and two mixtures of charged and uncharged lipids (Brethes, D., Dulon, D., Johannin, G., Arrio, B., Gulik-Krzywicki, T., Chevallier, J. 1986. Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 246:355–356) were analyzed in terms of four models of the membrane-water interface: (I) a smooth, negatively charged surface; (II) a negatively charged surface of lipid bilayer covered with an electrically neutral surface frictional layer; (III) an electrically neutral lipid bilayer covered with a neutral frictional layer containing a sheet of negative charge at some distance above the surface of the bilayer; (IV) an electrically neutral lipid bilayer covered with a homogeneously charged frictional layer. The electrophoretic mobility was predicted from the numerical integration of Poisson-Boltzmann and Navier-Stokes equations. Experimental results were consistent only with predictions based on Model-III with charged sheet about 4 nm above the bilayer and frictional layer about 10 nm thick. Assuming that the charge of the SR membrane is solely due to that on Ca⁺⁺-ATPase pumps, the dominant SR protein, the mobility data of SR and reconstituted SR vesicles are consistent with 12 electron charges/ATPase. This value compares well to the net charge of the cytoplasmic portion of ATPase estimated from the amino acid sequence (-11e). The position of the charged sheet suggests that the charge on the ATPase is concentrated in the middle of the cytoplasmic portion. The frictional layer of SR can be also assigned to the cytoplasmic portion of Ca⁺⁺-ATPase. The layer has been characterized with hydrodynamic shielding length of 1.1 nm. Its thickness is comparable to the height of the cytoplasmic portion of Ca⁺⁺-ATPase. Received: 15 June 1998/Revised: 8 October 1998     

Effect of vaginal distention on elastic fiber synthesis and matrix degradation in the vaginal wall: potential role in the pathogenesis of pelvic organ prolapse

Matrix metalloprotease (MMP) activity is increased in the postpartum vagina of wild-type (WT) animals. This degradative activity is also accompanied by a burst in elastic fiber synthesis and assembly. The mechanisms that precipitate these changes are unclear. The goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. Vaginal distention with a balloon simulating parturition resulted in increased MMP-2 and MMP-9 activity in the vaginal wall of nonpregnant and pregnant animals. This was accompanied by visible fragmented and disrupted elastic fibers in the vaginal wall. In nonpregnant animals, the abundant amounts of tropoelastin and fibulin-5 in the vagina were not increased further by distention. In contrast, in pregnant animals, the suppressed levels of both proteins were increased 3-fold after vaginal distention. Distention performed in fibulin-5-deficient (Fbln5(-/-)) mice with defective elastic fiber synthesis and assembly induced accelerated pelvic organ prolapse, which never recovered. We conclude that, in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. Distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. The finding that distention results in accelerated pelvic organ prolapse in Fbln5(-/-) animals, but not in WT, indicates that elastic fiber synthesis is crucial for recovery of the vaginal wall from distention-induced increases in vaginal protease activity.     

Regulation of elastolytic proteases in the mouse vagina during pregnancy, parturition, and puerperium

Recent evidence indicates that failure of elastic fiber assembly and synthesis is involved in the pathophysiology of pelvic organ prolapse in mice. It has been long been hypothesized that parturition-induced activation of proteases in the vaginal wall and its supportive tissues may contribute to pelvic organ prolapse in women. In this investigation, we determined the expression of matrix metalloproteases with elastase activity (matrix metalloproteinase [MMP] 2, MMP9, and MMP12) and their inhibitors in the vaginal wall of nonpregnant, pregnant, and postpartum mice. Data obtained using mRNA levels and enzyme activity measurements indicate that MMP2, MMP9, and 21- to 24-kDa caseinolytic serine proteases are regulated in vaginal tissues from pregnant and postpartum mice. Although suppressed during pregnancy and the early postpartum time period, MMP2 and MMP9 enzyme activities are increased after 48 h, a time when mRNA levels of protease inhibitors (tissue inhibitor of MMP2 [Timp2], cystatin C [Cst3], and alpha-1 antitrypsin [Serpina1]) are decreased. We conclude that recovery of the vaginal wall from pregnancy and parturition requires increased elastic fiber assembly and synthesis to counteract the marked increase in elastolytic activity of the postpartum vagina.     

Osmotically absorbed water preferentially enters the cutaneous capillaries of the pelvic patch in the toad Bufo marinus

Cutaneously absorbed water in anurans has two potential routes of movement from the skin interstitium into the body fluids, via the cutaneous capillaries and/or the lymphatic system. We investigated the lymphatic route at the pelvic patch skin under the influence of isoproterenol and arginine vasotocin in Bufo marinus by continuously aspirating lymph from the lymph sacs draining the pelvic patch while the animals absorbed water. Changes in body mass, lymph mass, and lymph osmolality were measured. If absorbed water entered the lymph space directly, we expected, relative to controls, (1) no difference in change in body mass, (2) lymph mass to be greater, and (3) lymph osmolality to be lower. None of these predictions were confirmed. We also tested the possibility that absorbed water was stored in the skin interstitium by measuring the surface density of pelvic skin immediately after it absorbed water. If water was stored, we expected the surface density of this skin to be greater than that of control skin. No difference in surface density was found. These results provide strong evidence that absorbed water does not directly enter the lymphatic system and is not stored in the skin. Consequently, osmotically absorbed water must enter via a transcapillary route.