Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.     

Prenatal exposure of male mice to diethylstilbestrol alter the expression of the lactotransferrin gene in seminal vesicles

We have previously isolated an estrogen-inducible secretory protein, lactotransferrin (LTF), and a cDNA to its messenger RNA from the uterus of mice. In this report we determined that the level of LTF mRNA is minimal in the seminal vesicles of normal mice. In contrast, expression of LTF mRNA in the seminal vesicles of developmentally estrogenized males was both constitutive and estrogen inducible. The results suggested that this alteration may be an example of atypical gene expression after hormonal manipulation early in development.     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

Molecular interactions between human lactotransferrin and the phytohemagglutinin-activated human lymphocyte lactotransferrin receptor lie in two loop-containing regions of the N-terminal domain I of human lactotransferrin.

Fluorescein isothiocyanate derivatization of the human lactotransferrin on Lys-264 inhibits the binding of the protein of human PHA-activated lymphocytes [Legrand, D., Mazurier, J., Maes, P., Rochard, E., Montreuil, J., & Spik, G. (1991) Biochem. J. 276, 733-738], indicating that part of the receptor-binding site is located in the N-terminal domain I of lactotransferrin. In the present study, a 6-kDa peptide (residues 4-52) was isolated from the N-terminal lobe of human lactotransferrin which inhibited the binding of the protein to its cell receptor. In addition, lactotransferrin was derivatized using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and sulfosuccinimidyl 6-((4'-azido-2'-nitrophenyl)amino)hexanoate (sulfo-SANPAH), two heterobifunctional reagents generally used for receptor-ligand cross-linking. The azide group of these two reagents was inactivated by photolysis, and only the succinimidyl ester group was allowed to react with lysine residues of the protein. The binding of the derivatized lactotransferrins to the human lymphocyte receptor was assayed. SASD, which binds to Lys-74, was able to inhibit the binding of lactotransferrin to the cell receptor, in contrast to Lys-281-binding sulfo-SANPAH. Molecular modeling showed the position of SASD, sulfo-SANPAH, and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask residues 4-6 and two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). The comparison of the primary and tertiary structures of human lactotransferrin and serotransferrin, which bind to specific cell receptors, shows that the above-mentioned regions, which are likely involved in protein-receptor interactions, possess specific structural features.     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

Estrogen induction of a small, putative K+ channel mRNA in rat uterus

Estrogen causes dramatic long-term changes in the activity of the uterus. Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes. The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources. It is, however, similar to structural motifs found in certain prokaryotic ion channels. The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment. These results support a critical role for regulation of ion channel expression by estrogen in the uterus.     

Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus

Populations of macrophages and neutrophils in the uterus are under the control of the female sex steroids estrogen and progesterone (P4). Their influx is induced by estrogen, while P4 can both stimulate and inhibit leukocyte influx depending on the timing of P4 with respect to estrogen. Regulation of leukocytes has been implicated in changes in uterine immune responses during the estrous cycle, pregnancy, and implantation. This work demonstrates that P4 given concurrently with estrogen to ovariectomized mice for 4 days antagonizes the ability of estrogen to recruit macrophages and neutrophils into the mouse uterus. Using progesterone receptor knockout (PRKO) mice, we show that this effect is dependent on progesterone receptors (PR). In the absence of PR, neutrophils recruited by estrogen were found to be degranulated, partially explaining the edema that is observed with long-term treatment of PRKO mice with estrogen and P4. Populations of B lymphocyte cells were shown to be unchanged by estrogen and P4 treatment in both wild-type and PRKO mice. The neutrophil chemotactic chemokine MIP-2 was examined for down-regulation by P4 but was found to be unaffected by hormonal treatment. Together, these observations demonstrate that PR has a strong anti-inflammatory role in the mouse uterus when estrogen and P4 are present together.     

Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus

Leukemia inhibitory factor (LIF) has been shown to be essential for the implantation of mouse blastocysts. The present study was designed to determine how LIF protein was hormonally regulated in rabbit and mouse uterus using immunohistochemistry. In unmated rabbits, LIF protein was at a low level in the uterine epithelium and glands, and up-regulated by progesterone alone or estradiol-17β and progesterone combined. Estradiol-17β alone had no apparent effect. In ovariectomized mice, the level of LIF protein was very low in the uterine epithelium and glands, and was up-regulated by estradiol-17β alone or estradiol-17β and progesterone combined. Progesterone alone had no apparent effect. These results suggest that LIF protein is differentially regulated in rabbit and mouse uterus by progesterone and estrogen, respectively. This would explain the high level of LIF protein observed in uterine epithelium and glands prior to blastocyst implantation in the two species with different hormonal requirements for implantation. © 1996 Wiley-Liss, Inc.     

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.     

Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen.

The effects of the steroid hormones estrogen and progesterone on peptidylarginine deiminase protein-L-arginine iminohydrolase, EC 3.5.3.15) levels in adult ovariectomized mouse uterus were studied. The amount of the enzyme in the uterus was considerably diminished by ovariectomy. When the mice were injected with a variety of estrogenic compounds, 17 beta-estradiol-3-benzoate, which was the most potent stimulator of uterine cell proliferation among the estrogens tested, dramatically elevated the enzyme formation of the uterus in a dose- and time-dependent fashion. Results of immunohistochemistry with the antiserum against mouse peptidylarginine deiminase demonstrated that the induction of the enzyme by the estradiol exclusively occurred at the luminal and glandular epithelia, corresponding with the previous findings in the normal estrous cycle. Furthermore, administration of the estradiol significantly increased the content of mRNA coding for peptidylarginine deiminase in uterus, indicating the evidence of regulation in pretranslation. On the other hand, progesterone alone did not restore the enzyme level of the ovariectomized mouse, but moderated the action of estrogen when given in concert with estrogen. Thus, the expression of peptidylarginine deiminase in luminal and glandular epithelia of mouse uterus is controlled by the amount of the steroid hormones estrogen and progesterone.     

Visualization of lactotransferrin brush-border receptors by ligand-blotting

The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120-128). In the present data, using 125I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 X 10(13) sites per mg of membrane proteins with an equilibrium constant of 1.2 X 10(6) M-1. Sodium dodecyl sulfate solubilization of the brush-border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with 125I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border.