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Endocrinology Vol. 140, No. 4 1852-1860
Copyright © 1999 by The Endocrine Society

ARTICLES

Restricted Spatiotemporal Expression of Lactoferrin during Murine Embryonic Development

Pauline P. Ward, Marisela Mendoza-Meneses, Biserka Mulac-Jericevic, Grainne A. Cunningham, Odila Saucedo-Cardenas, Christina T. Teng and Orla M. Conneely

Department of Cell Biology (P.P.W., M.M.-M., B.M.-J., G.A.C., O.S.-C., O.M.C.), Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030; and National Institutes of Environmental Health Sciences (C.T.T.), National Institutes of Health, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Orla M. Conneely, Department of Cell Biology, Room M513A, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: orlac{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lactoferrin is a member of the transferrin family of iron-binding glycoproteins. Lactoferrin is induced by estrogen in the mouse uterus during early pregnancy. However, the expression and function, if any, of lactoferrin in the preimplantation embryo during this developmental period has not been investigated. In the current study, the spatiotemporal expression of lactoferrin during murine embryogenesis was examined using in situ hybridization and immunohistochemical analyses. Lactoferrin expression was first detected in the 2–4 cell fertilized embryo and continued until the blastocyst stage of development. Interestingly, at the 16-cell stage, coinciding with the first major differentiation step in the embryo, lactoferrin messenger RNA (mRNA) is synthesized by the inner cells, whereas the protein is selectively taken up by the outside cells. This differential pattern of lactoferrin messenger RNA and protein localization continues until the blastocyst stage, with expression almost absent in the hatched blastocyst. Lactoferrin expression does not resume in the embryo until the latter half of gestation, where it is first detected in neutrophils of the fetal liver at embryonic day 11.5 and later in epithelial cells of the respiratory and digestive systems. Our results show that lactoferrin is expressed in a tightly regulated spatiotemporal manner during murine embryogenesis and suggest a novel paracrine role for this protein in the development of the trophoectodermal lineage during preimplantation development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LACTOFERRIN (Mr = 80 kDa) is a member of the transferrin family of nonheme iron-binding glycoproteins (1). Though lactoferrin displays extensive sequence and structural homology with members of the transferrin family, it is unique with respect to its expression and localization and also with regard to the pleiotropic functions that have been attributed to this protein. Lactoferrin is expressed and secreted primarily by epithelial cells (2, 3, 4). Highest levels of lactoferrin have been detected in the lactating mammary gland, where levels up to 6 g/liter have been detected in colostrum (5).

Lactoferrin is also present in tears and in nasal, salivary, bronchial, pancreatic, stomach, gastrointestinal, and genital secretions (2, 3, 6, 7, 8, 9). In addition, lactoferrin is expressed during the myelocytic stage of neutrophil development and is stored in the secondary granules of mature neutrophils, where it can be released into the bloodstream upon neutrophil activation and degranulation (10, 11, 12).

A diverse number of physiological functions have been proposed for lactoferrin, based on both in vitro and in vivo evidence. These functions include antimicrobial activity exerted by bacteriostatic (13, 14), bactericidal (15, 16, 17), and antiendotoxin (18) mechanisms, iron homeostasis (19, 20), cellular growth promotion (21, 22) and differentiation (23, 24), immunomodulatory activity (25, 26), and regulation of myelopoiesis (27, 28, 29). Specific and saturable receptors for lactoferrin have been identified on immune and nonimmune cell types, and these receptors may play an important role in modulating the pleiotropic functions of this protein (20, 30, 31, 32).

Lactoferrin has been shown to be differentially regulated by hormone in a tissue-specific manner. For example, lactoferrin expression is under the control of PRL (33) in the mammary gland; whereas, in the reproductive tract, the expression of this protein is inducible by the steroid hormone, estrogen (6, 34, 35). During the natural estrous cycle in the mouse, the level of lactoferrin correlates positively with circulating estrogen levels (36). Furthermore, lactoferrin is induced to high levels in the uterine epithelia during the first 2 days of pregnancy, after the preovulatory surge of estrogen (37). The protein returns to basal levels of expression by days 3–4, at the time of implantation corresponding to estrogen receptor down-regulation by progesterone released from the corpus luteum (38). This tight hormonal control of lactoferrin suggests that this protein may play an important physiological role in the uterus during early pregnancy. However, the expression pattern of lactoferrin, if any, during embryo development has not been investigated to date. To gain further insights into the function of lactoferrin during early gestation, we have examined the expression of this protein in the embryo using a combination of in situ hybridization, immunohistochemistry, and radiolabeled protein uptake studies. These analyses demonstrate that lactoferrin is expressed in a tightly regulated manner during murine embryo development and predict a novel paracrine role for this protein in the development of the trophoectodermal layer during murine preimplantation embryonic development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mice were housed in a 12-h light, 12-h dark cycle and in compliance with NIH and institutional guidelines.

Isolation of preimplantation mouse embryos
Wild-type 129sv/C57BL6 or ICR mice (Harlan Sprague Dawley, Inc., Houston, TX) were superovulated by an ip injection of 5 IU PMS gonadotrophin (Diosynth, Chicago, IL), followed 48 h later with 5 IU human CG (hCG, Pregnyl, Organon, West Orange, NJ) and placed with 129sv/C57BL6 stud males. The superovulated females were killed 1–4 days later, and the oviducts and/or uteri were flushed with M2 media (Sigma Chemical Co., St. Louis, MO) to obtain 1-cell to blastocyst stage embryos.

Isolation of postimplantation mouse embryos
Natural matings were set up between 129sv/C57BL6 mice, and the morning of detection of the vaginal plug was designated day 0.5 of pregnancy. At embryonic days E6–E18, pregnant mice were killed, and the embryos were isolated.

In situ hybridization analysis
Preimplantation embryos were fixed directly onto slides using 4% paraformaldehyde. Ovaries, oviduct, uteri, and postimplantation embryos were embedded in Tissue-Tek (Sakura Finetek U.S.A., Inc., Torrance, CA), frozen on dry ice, and fixed in 4% paraformaldehyde after sectioning. In situ hybridization analysis was performed using a 35S-labeled antisense mouse lactoferrin probe containing nucleotides 2008–2223 of the mouse lactoferrin complementary DNA (6) on 30-µm tissue sections or on embryo whole mounts (39). Sections were counterstained using hematoxylin, dehydrated through graded ethanol solutions, cleared in xylenes, and coverslipped for bright- or dark-field microscopy. Images were acquired using a Zeiss Axioscope microscope (Carl Zeiss, Thornwood, NY) coupled with a Hamamatsu C5810CCD camera (Hamamatsu Corp, Bridgewater, NJ). Images were processed using Adobe Photoshop 4.0 (Adobe Systems, Inc., San Jose, CA).

Immunohistochemical localization of lactoferrin in preimplantation and postimplantation embryos
Preimplantation embryos were fixed in 4% paraformaldehyde, and lactoferrin protein was localized using the previously well-characterized rabbit antilactoferrin polyclonal antibody (34), followed by fluoroisothiocyanate (FITC) or Texas Red conjugated secondary IgG (Cappel, Durham, NC). E-cadherin was detected using a rat monoclonal IgG (Catalog no. U-3254, Sigma Chemical Co.), followed by FITC-conjugated antirat IgG (Cappel). Confocal microscopy was performed using a Molecular Dynamics, Inc. probe 2001 laser-scanning microscope. Postimplantation embryos were fixed in Bouins solution for 2–24 h, depending on the size of the embryo. After fixation, the embryos were washed repeatedly in 70% ethanol and embedded in paraffin. Tissue sections (5-µm) were mounted on microscope slides and were deparaffinized and rehydrated through a graded series of ethanols. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide/methanol. The sections were incubated for 1 h in blocking solution (10% normal goat serum), followed by incubation for 3 h with rabbit polyclonal antiserum directed against mouse lactoferrin (1:1000). Sections were incubated next with a biotinylated goat antirabbit secondary antibody (1:1000). Immunoreactivity was detected using Streptavidin-Peroxidase (Zymed Laboratories, Inc., San Francisco, CA) and DAB (3,3'-diaminobenzidine) as substrate chromogen (Sigma Chemical Co.). Sections were counterstained using hematoxylin or a combination of hematoxylin and eosin, dehydrated through graded ethanol solutions, cleared in xylenes, and coverslipped for bright-field microscopy. Images were acquired and processed as described above. Neutrophils were detected, essentially as described above, using a 1:100 dilution of purified rat antimouse neutrophil monoclonal antibody (clone 7/4, catalog no. AMU0021, Biosource International, Camarillo, CA), followed by incubation with a 1:200 dilution of biotinylated goat antirat antibody (catalog no. BA-9400, Vector Laboratories, Inc., Burlingame, CA).

Lactoferrin binding analysis
Day-2.5 (8-cell) embryos were collected and cultured for 24 h. Lactoferrin binding was performed as described previously (40). Briefly, the zona pellucida was removed by incubation with 0.5% Pronase (Boehringer Mannheim, Indianapolis, IN) in M2 media (Speciality Media, Lavallette, NJ). Embryos were washed several times in KSOM medium (PGC Scientific, Gaithersburg, MD) and cultured for 2 h in M16 medium. The embryos were incubated in 50-µl microdrops of PBS/0.1% BSA containing 4 x 10-8 M iron-saturated recombinant mouse 125I-lactoferrin (DuPont NEN, Boston) at 4 C (see Fig. 3Go, A and C) or 37 C (see Fig. 3BGo) for 1 h in the presence or absence of a 250-fold excess of unlabeled iron-saturated recombinant lactoferrin. The embryos were then washed 3 times in cold PBS/0.1% BSA and fixed directly on charged microscope slides in cold 4% paraformaldehyde for 15 min. The slides were dehydrated through ascending concentrations of alcohol, dipped in Kodak NBT-2 emulsion (Eastman Kodak Co., New Haven, CT), exposed for 7 days, and developed and stained in hematoxylin.



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  		Figure 3. 125I-lactoferrin uptake in the preimplantation embryo. Autoradiograph of 16- to 32-stage embryos incubated with 125I-lactoferrin at 4 C (A and C) or 37 C (B) in the absence (A and B) or presence (C) of a 250-fold excess of unlabeled lactoferrin. Scale bar, 10 µM; ICM, inner cell mass; TE, trophoectoderm.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of lactoferrin during preimplantation development
The preimplantation phase of murine embryogenesis is characterized by the development of a fertilized egg to a fully expanded blastocyst. Though the first few cleavage events result in the formation of nonpolar equipotent blastomeres, the transition from the 8- to the 16-cell stage marks the first overt differentiation step in the embryo. At this developmental stage, the embryo compacts and polarizes, resulting in the appearance of two distinct cell lineages: the inside cells (which subsequently give rise to the inner-cell mass); and the outer cells (which give rise to the trophoectodermal layer) (41, 42, 43). Subsequent cell divisions result in the development of an expanded 128-cell blastocyst that hatches from the zona pellucida and implants into the uterine wall at embryonic day 4 (44, 45). To determine whether lactoferrin is expressed during this period of mouse development, in situ hybridization was performed using a 35S-labeled lactoferrin antisense riboprobe. The results from this analysis are shown in Fig. 1Go. Lactoferrin messenger RNA (mRNA) is absent from the developing oocyte (Fig. 1Go, A and D), and transcripts were first detected at the 2–4 cell stage in the fertilized embryo (Fig. 1Go, B and E), where it continued to be expressed in all blastomeres until the 8-cell stage of embryonic development. At the 8- to 16-cell stage, however, staining for lactoferrin mRNA became concentrated in the inside cells but was absent from the outer trophoectodermal cells (Fig. 1Go; C, F, G, and J). This mRNA expression pattern continued in the inner cells, albeit at decreased levels, through the 32-cell stage (Fig. 1Go, H and K). However, expression of lactoferrin mRNA is nearly absent in the hatched blastocyst at the periimplantation stage (Fig. 1Go, I and L). Sections from the oviduct, which expresses high levels of lactoferrin (36), were used for antisense (Fig. 1MGo) and sense controls (Fig. 1Go, N and O) to demonstrate probe specificity.



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  		Figure 1. In situ hybridization analysis of lactoferrin expression in the developing oocyte and preimplantation embryo. Embryos were hybridized with a specific 35S-labeled antisense mouse lactoferrin probe. A–C, G–I, and N, Bright-field illumination; D–F, J–M, and O, dark-field illumination; A and D, developing oocyte; B and E, 2- to 4-cell embryo; C and F, 8- to 16-cell embryo; G and J, 16-cell embryo; H and K, 32-cell embryo; I and L, hatched blastocyst; M, oviduct, dark-field illumination; N and O, light-field (N) and dark-field (O) illumination of oviduct using sense control; scale bars, 20 µM (A–L) and 50 µM (M–O).

 
To gain further insights into the specific function of lactoferrin at this developmental stage, the protein localization in the preimplantation embryo was examined using an indirect immunofluorescence assay. Analysis of 8-cell embryo for lactoferrin immunoreactivity demonstrated that the protein is located extracellularly in the perivitelline space separating the blastomeres from the zona pellucida (Fig. 2AGo). However, as development progresses through the 16- to 32-cell stages (Fig. 2Go, B and C), lactoferrin immunofluorescence redistributes in the embryo and seems to be selectively localized within the cells of the outer trophoectodermal lineage in a pattern distinctly different from that observed for the lactoferrin mRNA. Double immunostaining with antibodies directed against the cell adhesion protein, E-cadherin, confirmed the specific localization of lactoferrin exclusively to the outer cells (Fig. 2DGo). Nonimmune controls did not show any staining from the same stage embryos (Fig. 2Go, E–H).



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  		Figure 2. Localization of lactoferrin protein in the preimplantation embryo. Lactoferrin protein was localized in preimplantation embryos using an indirect immunofluorescence assay using antilactoferrin IgG, followed by an FITC-conjugated secondary antibody (green). A, 8-cell embryo; B, 16-cell embryo; C, 32-cell embryo; D, double immunostaining of blastocyst embryo using antilactoferrin IgG, followed by a Texas red conjugated secondary antibody (red) and anti-E-cadherin, followed by an FITC-conjugated secondary antibody (green). Control sections incubated with nonimmune serum, in place of primary antibody, are shown on the bottom panel for each embryo stage (E–H). Scale bars, 10 µM (A–C) and 20 µM (D–H).

 
To confirm that the intracellular lactoferrin results from uptake rather than de novo synthesis in trophoectodermal cells, 16- and 32- to 64-cell embryos were incubated with 125I-lactoferrin, in the absence of the zona pellucida, and the distribution of the labeled protein was followed. As predicted by the immunofluorescence assays, binding and internalization of lactoferrin was observed in the outer cells of the 16-cell embryo (Fig. 3AGo) and in the trophoectoderm, but not the inner cell mass of the 32- to 64-cell blastocyst (Fig. 3BGo). This binding and internalization was blocked by incubation with a 250-fold excess of cold unlabeled lactoferrin (Fig. 3CGo). These results suggest that the outside cells selectively expresses receptor binding sites for lactoferrin that are capable of selectively internalizing this protein.

Lactoferrin expression coincides with the onset and diversification of myelopoiesis in the postimplantation embryo
To determine whether embryonic lactoferrin expression is restricted to the preimplantation stage of development, we analyzed the spatiotemporal pattern of lactoferrin mRNA expression during post implantation. The results of these in situ hybridization analyses are shown in Figs. 4–7GoGoGoGo. Lactoferrin mRNA was not detected in the postimplantation embryo until embryonic day 11.5, when a subpopulation of cells in the fetal liver stained positively for lactoferrin at the time of onset of myelopoiesis in this organ (Fig. 4AGo). The number of positively stained cells was highest at embryonic day 15.5, coincident with maximal hematopoietic activity of the embryonic liver (Fig. 4BGo), and were still detected (but decreased in number) by late gestation, at E17.5 (Fig. 4CGo). To determine whether lactoferrin was localized in the neutrophils of the liver, immunohistochemical analysis was performed using serial sections labeled with a murine lactoferrin-specific antibody (E) or a murine neutrophil-specific antibody (G). Consistent with the mRNA results, a subpopulation of cells immunoreactive for lactoferrin was detected in the liver (Fig. 4EGo). Hepatocytes and erythrocytes are clearly devoid of immunoreactivity. Analysis of immunoreactive cells at higher magnification showed that the majority of the lactoferrin-staining cells had a multilobed nucleus and granular cytoplasm and were similar morphologically to neutrophil-staining cells (Fig. 4Go, E and G, insets). Additional cell types in the liver also seem to be positive for lactoferrin; and, while these cells may be macrophages, which contain receptors for lactoferrin (32), their exact identity will require further analysis. Control liver sections, incubated with sense lactoferrin riboprobe (Fig. 4DGo) or nonimmune serum, in immunohistochemistry, did not show any staining (Fig. 4Go, F and H).



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  		Figure 4. In situ hybridization and immunochemical localization of lactoferrin in the fetal liver. A–C, In situ hybridization analysis using a specific 35S-labeled antisense mouse lactoferrin probe; A, E11.5 liver; B, E15.5 liver; C, E17.5 liver; D, E17.5 liver using a control sense 35S-labeled mouse lactoferrin probe; E, immunohistochemical analysis of cross-section of fetal liver (E16.5) incubated with a polyclonal antibody directed against mouse lactoferrin; E, inset, high-power magnification (116x) of fetal liver, showing a neutrophil with multilobed nucleus, which is staining positive for lactoferrin; F, negative control section of liver, at 16.5, incubated with nonimmune serum; G, immunohistochemical analysis of a cross-section of fetal liver (E16.5) incubated with a neutrophil specific (clone 7/4) antibody; G, inset, high-power magnification (116x) of neutrophil; H, neutrophil negative control section of liver, at 16.5, incubated with nonimmune serum; A–D, dark-field illumination; E–H, bright-field illumination; scale bars, 200 µM (A–D) and 50 µM (E–H).

 


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  		Figure 5. In situ hybridization and immunohistochemical analysis of lactoferrin localization in the fetal bone marrow and spleen. In situ hybridization analysis: Sagittal sections were obtained from embryos at day 17.5 of gestation; A and D, dark-field illumination of in situ hybridization of fetal bone marrow (A) and spleen (D) using a specific 35S-labeled antisense mouse lactoferrin probe. Immunohistochemical analysis: B and C, immunohistochemical analysis of serial sections (E16.5) of fetal bone marrow incubated with either mouse lactoferrin antibody (B) or mouse neutrophil antibody (C). E and F, immunohistochemical analysis of serial sections (E16.5) of fetal spleen incubated with either mouse lactoferrin antibody (E) or mouse neutrophil antibody (F). Insets, high-power magnification (116x) of a neutrophil; scale bars, 50 µM (A and D–F) and 30 µM (B and C).

 


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  		Figure 6. In situ hybridization and immunohistochemical localization of lactoferrin in the digestive tract. Light- (A–C) and dark-field (E–G) illumination of sagittal sections from day-17.5 embryos incubated with a specific 35S-labeled antisense mouse lactoferrin probe. A and E, oral cavity; B and F, esophagus; C and G, stomach. Immunohistochemical analysis of fetal stomach incubated with a polyclonal antibody directed against mouse lactoferrin (H) or nonimmune serum control (D). Scale bars, 50 µM (A, B, E, and F) and 200 µM (C, D, G, and H).

 


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  		Figure 7. In situ hybridization analysis of lactoferrin in the respiratory system. Sagittal sections were obtained from day-17.5 embryos and incubated with a specific 35S-labeled antisense mouse lactoferrin probe. A, nasopharynx; B, nasal cavity; C, oropharynx; D, trachea; scale bars, 50 µM.

 
During late fetal development, definitive myelopoiesis diversifies from the liver to the spleen and bone marrow at embryonic day 16.5 (46). Corresponding to this diversification, lactoferrin mRNA was also detected in the neutrophils in the fetal bone marrow and spleen at embryonic days 16.5–17.5 (Fig. 5AGo and D). Immunohistochemical analysis, using serial sections incubated with a lactoferrin- or neutrophil-specific antibody, showed a similar number of cells immunoreactive for lactoferrin and neutrophils in both the bone marrow (Fig. 5Go, B and C) and spleen (Fig. 5Go, E and F). Furthermore, analysis of these cells at high power magnification confirmed the identity of the lactoferrin-positive cells as neutrophils (Fig. 5Go; B, C, E, and F, insets). No lactoferrin mRNA transcripts were detected in the thymus, the site of lymphoid cell development (results not shown).

Lactoferrin mRNA expression in secretory epithelial cells
A third distinct spatiotemporal wave of lactoferrin mRNA expression was observed during late gestation, from embryonic days 16–17 onwards. This latter pattern corresponded to the onset of lactoferrin expression in secretory epithelial cells that is widely observed in the adult (2, 3, 8). These included the digestive system, where lactoferrin mRNA transcripts were restricted to the upper part of the developing digestive tract in epitheliocytes of the oral cavity (Fig. 6Go, A and E), esophagus (Fig. 6Go, B and F), and stomach (Fig. 6Go, C and G). The specific localization of lactoferrin protein in gastric epithelial cells was confirmed using immunohistochemical analysis (Fig. 6HGo). No mRNA transcripts were detected in the embryonic intestine (results not shown). At the same embryonic stage, lactoferrin mRNA was also detected in the respiratory system, where it was localized in the epithelial cells of the nasopharynx, nasal cavity, oropharynx, and trachea (Fig. 7Go, A–D). No mRNA transcripts for lactoferrin were detected in the embryonic lung (results not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that lactoferrin is expressed during murine embryogenesis in three distinct spatiotemporal patterns. The first is initiated in the fertilized 2- to 4-cell embryo and is restricted to the preimplantation phase of early gestation. The second two patterns do not occur until the latter half of gestation and are localized to the neutrophils after the onset of myelopoiesis and appearance of epitheliocytes of the developing respiratory and digestive systems.

Most interestingly, however, we show that lactoferrin is expressed in a restricted manner in the preimplantation embryo. Comparison of the expression pattern of this protein shows a striking correlation between embryo and maternally expressed lactoferrin during early gestation. Lactoferrin is known to be up-regulated in the maternal endometrium on days 1 and 2 of pregnancy (37), and this up-regulation is associated with a synchronous high level of expression of the mRNA at the 2- to 16-cell stage embryo. Further, cessation of lactoferrin expression in the maternal endometrium on day 3.5 (37) is paralleled by the down-regulation of lactoferrin expression in the periimplantation embryo. It has been well documented that the control of lactoferrin expression in the uterus during this period is regulated by estrogen and mediated by estrogen receptor-dependent activation of the mouse lactoferrin promoter (6, 34, 36, 47). Further, estrogen-induced expression of lactoferrin has been shown to be inhibited by progesterone acting via its nuclear receptor (48). Although the mechanism by which embryonic lactoferrin expression may be synchronously regulated has yet to be determined, both estrogen and progesterone receptors have been detected in the preimplantation embryo (49, 50), raising the possibility that estrogen and progesterone also play a direct role in regulation of expression of embryonic lactoferrin.

Furthermore, we have shown that the localization of lactoferrin is restricted in the preimplantation embryo. In the undifferentiated embryo, lactoferrin mRNA is expressed equally in all blastomeres, whereas the protein is secreted extracellularly into the perivitelline space. However, at the 16-cell stage, coinciding with the first overt differentiation in the embryo, the mRNA is expressed exclusively in the inside cells, whereas the protein redistributes and is selectively taken up in a paracrine manner by the outer polar cells of the trophoectodermal lineage. This expression pattern continued until the blastocyst stage, with almost no lactoferrin mRNA detected in the hatched blastocyst. The lack of localization or uptake of lactoferrin protein in the inside cells, together with the lack of observed expression of this protein until the second half of gestation, suggest that this protein does not play a role in the development of embryo-derived tissues at this time and that its function is restricted to the development of the extraembryonic preimplantation trophoectoderm during the first half of gestation. In the neonate and adult, lactoferrin has been postulated to be an important regulator of cellular growth (21, 22) and differentiation (23, 24). However, the mechanism of action, and possible involvement of receptors in the development of the trophoectodermal cells by lactoferrin, remains to be established.

The latter two patterns of lactoferrin expression are consistent with the well-documented reports on the expression and localization of this protein in neutrophils and as a primary component of epithelial secretions in the neonate and adult, where it plays an important role in host defense (2, 3, 8, 10). Interestingly, though lactoferrin in hematopoietic cells seems to be localized exclusively in neutrophils in the adult, our results in fetal liver indicate that additional hematopoietic cells may also express or internalize lactoferrin during fetal hematopoiesis. Additional analysis will be required to identify the specific cells in which lactoferrin is localized and to establish the role of this protein in hematopoiesis in the embryo. Finally, the observation that lactoferrin mRNA is expressed throughout the epithelium of the upper digestive and respiratory tracts during early development suggests that the protein may play a role in proliferation and/or differentiation of the primitive digestive and respiratory tracts.

In summary, we demonstrate, for the first time, the spatiotemporal expression of lactoferrin during murine embryogenesis. We show that lactoferrin is expressed in a tightly regulated manner during embryonic development, with expression of this protein limited to the preimplantation embryo, postimplantation neutrophils, and epithelial cells of the developing digestive and respiratory tract. Furthermore, we provide evidence to support a novel paracrine role for lactoferrin in the preimplantation embryo in the selective development of the trophoectodermal lineage.

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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