Secretion and Immunolocalization of Retinol-Binding Protein in Bovine Conceptuses During Periattachment Periods of Early Pregnancy

Journal of Reproduction and Development, Vol. 48, No. 4, 2002

—Original—

Secretion and Immunolocalization of Retinol-Binding Protein in Bovine Conceptuses during Periattachment Periods of Early Pregnancy

Kaung Huei LIU1)

1)Department of Veterinary Science, National Chiayi University, Chiayi , Taiwan 300, Republic of China

Abstract. The purpose of the study was to determine and compare the secretion of RBP by bovine spherical, elongating and filamentous conceptuses, and to identify the cellular location of RBP in developing conceptuses by immmunocytochemistry. Bovine conceptuses were removed from the uterus between days 13 and 22 of pregnancy. Events of early bovine embryonic development were observed. The conceptuses underwent a transformation from a spherical to a filamentous morphology during the periattachment period of placentation. Isolated conceptuses were cultured in a modified minimum essential medium in the presence of radiolabeled amino acids. Presence of retinol-binding protein (RBP) in culture medium was determined by immunoprecipitation using bovine placental anti-RBP serum. Presence of immunoreactive RBP in detectable quantities in spherical blastocyst (day 13) culture medium was evident. Increased amounts of RBP were clearly detected in cultures on days 14 and 15, the time of elongating conceptuses. RBP was abundant in cultures on day 22, when the conceptuses were filamentous. Cellular sources of RBP in day 15 and 22 conceptuses were determined by immunocytochemistry with anti-RBP serum. Strong immunoreactive RBP was localized in trophectoderm of day 15 conceptuses, and in epithelial cells lining the chorion and allantois of day 22 conceptuses. RBP originating from the conceptus may serve to transport retinol locally from the uterus to embryonic tissues. Secretion of RBP by spherical blastocysts (day 13) suggests that retinol is needed for initiating the early events of embryonic development. Increased secretion of RBP on days 14 and 15, the time of elongating conceptuses, was coincident with the timing of rapid trophoblast hyperplasia and remodeling. Greater secretion of RBP by the filamentous conceptus (day 22) coincided with the timing of differentiation of the chorion and allantois as well as growth of the embryo proper. Production of RBP by the developing conceptuses may be indicative of an important role for conceptus RBP in the transport, availability and metabolism of retinol during early pregnancy. Key words: Bovine, Conceptus, Chorion, Allantois, Vitamin A (J. Reprod. Dev. 48: 423–430, 2002)

n bovine, communication between the recognition of pregnancy, occurs between days 16– developing conceptus and maternal tissues is 17 of pregnancy in cows [1, 2]. The major classes of essential for a successful pregnancy. Presence of compounds that may serve as signals for feto- conceptuses must be signaled before placentation maternal interactions include steroids, prostanoid, begins to prevent the regression of the corpora and proteins [3–5]. In vitro synthesis and release of lutea. The phenomenon, known as maternal proteins by intact bovine conceptuses between days 17 and 38 of pregnancy have been Accepted for publication: April 5, 2002 424 LIU eletrophoretically characterized [6]. Bovine Materials and Methods maternal recognition of pregnancy is mediated by conceptus secretory protein, known as bovine Materials trophoblast protein-1 (bTP-1). This protein has Eagle’s minimal essential medium (MEM) and high DNA sequence a class of interferon-α [7] and other supplies for tissue culture were purchased possess antiviral activities characteristic of from GIBCO, Grand Island, NY, USA. [35S]- interferon [8]. methionine and [3H]-leucine were obtained from Bovine TP-1 is a major secretory product of New England Nuclear, Boston, MA, USA. X-Omat conceptuses between days 15–24 in cattle [9] and AR film was a product of Eastman Kodak, can influence uterine prostaglandin metabolism Rochester, NY, USA. Reagents for [10], possibly by inducing an inhibitor of immunoperoxidase staining were obtained from prostaglandin F2α synthesis and prolonging the BioGenex, San Ramon, CA, USA. Supplies for lifespan of the corpus luteum [11]. Production of polyacrylamide gel electrophoresis and immune bTP-1 by the intact conceptus diminishes after day complex precipitation were as described by Lifsey 24 of pregnancy [6, 9], when another discrete 21 et al. [18]. All other chemicals were of reagent kDa protein, designated 3B3, synthesized by bovine grade or better and were products of Sigma placental membranes (chorion and allantois) Chemical Co, St. Louis, MO, USA. becomes prominent [12]. Recently 3B3 has been identified as a placental retinol-binding protein In vitro culture of conceptuses (RBP) by N-terminal amino acid analysis [13]. In Modified MEM with 20 µCi/ml [35S]-methionine serum, RBP is the specific transport protein for and 20 µCi/ml [3H]-leucine were prepared. Cyclic retinol (vitamin A alcohol) [14]. The bovine cows were inseminated on the day of estrus (day 0). placentation is classified as epitheliochorial type On days 13 (n=8), 14 (n=8), 15 (n=5) and 22 (n=4) [15]. During the periattachment period the conceptuses were flushed out of uteri under sterile conceptus is dependent upon uterine secretions for conditions. Eight day 13 spherical blastocysts (0.4– nutritional need. The nutrients including 1.2 mm in diameter) were pooled and cultured in 2 carbohydrates, minerals, proteins and vitamins, are ml modified MEM. Eight day 14 conceptuses (8–9 × necessary for embryonic differentiation, and 1 mm) were divided into four groups. Each group subsequent development and growth [15, 16]. containing two conceptuses were cultured in 2 ml Shortly after hatching the bovine blastocyst enters a modified MEM. Day 15 conceptuses (16–18 × 1 phase of rapid growth and development [16]. The mm) were transferred separately to individual bovine conceptus undergoes a transformation from sterile Petri dishes containing 2 ml modified MEM. spherical to filamentous morphology during Each piece (5–6 × 3 mm cutting) of day 22 periattachment period. Therefore, the period of conceptuses was transferred separately to sterile periattachment is critical for establishment and Petri dishes containing 2 ml modified MEM. maintenance of pregnancy. Since retinol plays Culture conditions were as described previously important roles in cell differentiation and [6]. Conceptuses were incubated at 37 C on a embryonic morphogenesis [17], it is likely that RBP rocking platform in a gaseous atmosphere of 50% may also be a secretory product of spherical, O2, 45% N2, and 5% CO2 (by volume). After 36 h, elongating and filamentous conceptuses. RBP culture was terminated by centrifuging at 12,000 × originating from the conceptus may serve to g for 15 min at 4 C to separate tissue from medium. transport retinol locally from the uterus to Conditioned medium samples were dialyzed embryonic tissues. The goals of this study were to against 10 mM Tris-HCl buffer, pH 7.6. determine and compare the secretion of RBP by bovine spherical, elongating and filamentous Immunoprecipitation conceptuses, and to identify the cellular location of Anti-bovine placental RBP antiserum (50 µl) was RBP in developing conceptuses by added to 1 ml of 35S- and 3H-labeled conceptus immmunocytochemistry. tissue culture medium and incubated over night at 4 C. Immune complex was collected onto protein-A Sepharose CL-4B in 40 mM Tris-HCl (pH 7.5). Immune complexes were solublized by boiling at RBP IN BOVINE CONCEPTUS 425

100 C for 5 min. Proteins contained in the Results supernatant were analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel Early bovine embryonic development electrophoresis (1D-SDS-PAGE). One-dimensional Events of early bovine embryonic development SDS-PAGE was performed according to the are shown in Fig. 1. The day 13 blastocyst was a method of Laemmli [19] in 12.5% (w/v) small spherical structure (0.4–1.2 mm in diameter). polyacrylamide gels and 5% of stacking gel. The spherical blastocyst consisted of a fluid-filled Following electrophoresis, Coomassie blue R-250- cavity and the surrounding trophectoderm (Fig. stained gels were dried after impregnation with 1 1A). On day 14 the blastocyst began to elongate (8– M sodium salicylate [20]. Fluorographs were 9 × 1 mm) (Fig. 1B, arrowhead). Location of the prepared and radiolabeled proteins were detected future embryo at the center of the day 15 conceptus using Kodak X-ray film. (16–18 × 1 mm) was observed (Fig. 1B, arrow). Over the next 7 days the conceptus underwent a Immunocytochemistry tremendous growth resulting in a filamentous Days 15 and 22 conceptus tissues were structure (200 × 3 mm) (Fig. 1C). By day 45, the immersion-fixed in Bouin’s solution for 2 h and differentiated extraembryonic membranes washed with water. Tissues were dehydrated and (chorion, allantois and amnion) and the embryo embedded in paraffin. Tissue sections (6 µm thick) proper were observed. Amnion contained amniotic were prepared. A modified immunoperoxidase fluid, in which the fetus was suspended. The method was used to detect RBP in these issues. allantois carried blood vessels to connect the fetus Briefly, it consisted of the following steps: with placental circulation. The outermost (a) Sections were completely covered with 3% membrane was the chorion. The allantois, which hydrogen peroxide and incubated for 10 min at was filled with allantoic fluid, fused with the room temperature (RT, 20–23 C) to block chorion to form the chorioallantois (Fig. 1D). endogenous peroxidase activity. (b) After rising well with phosphate buffered Immunoprecipitation of RBP saline (PBS), sections were treated with 1% bovine Immuno-complex precipitation with anti-RBP serum albumin for 20 min at 37 C. Sections were serum, followed by 1D-PAGE and fluorography blotted with paper to remove excess liquid. was used to identify RBP in bovine days 13–22 (c) Anti-RBP serum raised against RBP purified conceptus culture medium (Fig. 2). The presence of from bovine allantoic membrane culture medium the 21 kDa protein (RBP) in detectable quantities in and pre-immune serum (negative control) [13] day 13 spherical blastocyst culture medium was were prepared (1:500 dilution in PBS). After evident (lane A). Immunoprecipitated bands (21 blotting the slides, anti-RBP antiserum or non- kDa) were clearly detected in culture medium on immune serum were applied to sections for 30 min days 14 (lane B) and 15 (lane C), the time of at 37 C. elongating conceptuses. Increased intensity of the (d) After blotting the slides, the link antibody immunoprecipitated band was observed in day 22 (anti-immunoglobulin serum) was applied to filamentous conceptus culture medium (lane D). sections for 20 min at 37 C. Lane E, the control for lane C, showed only faint (e) Sections were washed with PBS and bands with molecular weights of 40,000–43,000. incubated in peroxidase-antiperoxidase solution in Non-immune serum did not react with the 21 kDa PBS with carrier protein (labeling antibody ) for 10 protein in day 15 conceptus culture medium (lane min at 37 C. E). Controls for lanes A, B and D were similar to (f) Sections were then washed with PBS and lane E (not shown). covered with 5% hydrogen peroxide for 10 min at RT. Immunolocalization of RBP (g) Finally, sections were covered with substrate Cellular location of RBP in day 15 and 22 solutions (aminoethylcarbazole) for 10–20 min at conceptuses was determinated by RT. Then they were washed with PBS, mounted immunocytochemistry using anti-RBP serum. with glycerol and examined under a microscope. Immunostaining RBP was observed in trophectoderm of the day 15 conceptus (Fig. 3A). 426 LIU

Fig. 1. Early embryonic development of bovine conceptuses: day 13 spherical (A), day 14 expanding (B, arrowhead), day 15 elongating (B, arrow), day 22 filamentous (C), and day 45 differentiated conceptuses (D).

No immunostaining was observed in the control section (Fig. 3B). Day 22 conceptus tissue sections showed that intensive immunostaining for RBP was detected in cells of the chorion (Fig. 4A, large arrow) and allantois (Fig. 5A, black arrow). Location of nuclei in cells was observed (Fig. 4A, small arrow; Fig. 5A, white arrow). No immunostaining of the control sections was evident (Figs. 4B and 5B).

Fig. 2. Identification of RBP secreted by days Discussion 13–22 conceptuses. Proteins in culture medium conditioned by conceptuses were immunoprecipitated with anti- In primates and rodents, blastocysts become RBP serum, and analyzed by 1D-SDS- embedded in the uterine mucosa by phagocytizing PAGE and fluorography. Exposure and digesting through the uterine luminal time was six weeks. Lane A: Day 13 spherical conceptus; Lane B: Day 14 epithelium. The blastocysts are thus encapsulated expanding conceptus; Lane C: Day 15 beneath the uterine luminal surface [21]. In elongating conceptus; Lane D: Day 22 contrast, the bovine blastocyst never becomes filamentous conceptus; Lane E: embedded in the uterine wall in the same manner control for lane C. as those of primates and rodents. The bovine conceptus remains within the uterine lumen in early pregnancy. The chorioallantois contacts and adheres to the uterine epithelium for placental RBP IN BOVINE CONCEPTUS 427

Fig. 3. Immunocytochemical localization of RBP in a conceptus of day 15 of pregnancy. Strong immunostaining was seen in trophoblast cells in the trophectoderm (A) when anti-bovine placental RBP antiserum was applied. Specific immunostaining was absent in the control section (B) when non-immune serum was applied. Panel A, × 60; Panel B, × 60.

Fig. 4. Immunocytochemical localization of RBP in the chorion of day 22 of pregnancy. Strong immunostaining was seen in epithelial cells of the chorion (A, large arrow) when anti-bovine placental RBP antiserum was applied. Location of the nucleus is indicated by a small arrow. Specific immunostaining was absent the in the control section (B) when non- immune serum was applied. Panel A, × 180; Panel B, × 180. attachment in the later stages of pregnancy [22]. structure [16]. During day 14–15 the spherical In bovine, the periattachment period is between blastocyst underwent logarithmic growth and days 4 and 22 of pregnancy. By day 4–5, the entered the elongation phase as observed in the fertilized egg develops to the 8–16 cell morula stage present study. Over the next week the conceptus and moves from the oviduct to the uterus [3]. The underwent tremendous growth, resulting in a compact morula develops into a fluid-filled filamentous structure, and by day 22, the blastocyst on day 6–8 [23]. On day 11, the extraembryonic membranes (chorion and allantois) blastocyst containing an inner cell mass hatches had begun to differentiate. By day 45 the from the zona pellucida. A single layer of chorioallantois had undergone a dramatic trophectoderm surrounds the spherical blastocyst expansion, and the embryonic tissues had formed [16]. The whole of the embryo proper is derived the fetus proper (observations of the present study). from the inner cell mass [16]. The trophectoderm is Day 13–22 conceptuses acquired the abilities of the precursor of the placenta and the earliest protein synthesis and secretion of RBP as component of the system of the extraembryonic demonstrated by immunoprecipitation with anti- 428 LIU

Fig. 5. Immunocytochemical localization of RBP in the allantois of day 22 of pregnancy. Strong immunostaining was seen in epithelial cells of the allantois (A, black arrow) when anti-bovine placental RBP antiserum was applied. Location of the nucleus is indicated by a white arrow. Specific immunostaining was absent in the control section (B) when non-immune serum was applied. Panel A, × 180; Panel B, × 180.

RBP serum in this study. In our earlier studies, rapid trophoblast hyperplasia and remodeling. expression of RBP was identified in a day 13 The conceptus elongates throughout the ipsilateral tubular conceptus [24]. Although day 13 spherical uterine horn on day 15 reaching the contralateral conceptuses secreted relatively low levels of RBP, horn by day 18 [16]. Greater production of RBP by the present study further demonstrated the ability the day 22 filamentous conceptus, observed in the of RBP production by day 13 spherical blastocysts present study, is coincident with the timing of (prior to expansion). Once secretion of RBP by day differentiation of the chorion and allantois as well 13 spherical conceptuses had been initiated, day as growth of the embryo proper. 14–15 conceptuses were much more active in RBP Retinoids (vitamin A and its analogs) are secretion. Secretion of conceptus RBP continuously essential for growth and differentiation of epithelial increased through day 13–22 of pregnancy. The tissues [25]. In addition, retinoids influence the cellular source of RBP in day 13–15 conceptuses gene expression that regulates many proteins was the trophoblast cell. The cellular location of associated with the formation of cytoskeleton and RBP in day 22 conceptuses was identified as being extracellular matrix [26–29]. Retinoic acid, a in the chorion and allantois, observed in the present metabolite of retinol, is known to be involved in study. These results clearly demonstrated that RBP morphogenesis of the anteroposterior axis pattern was synthesized and secreted by bovine of the chick embryo [30–33], and the nervous conceptuses in the periattachment period. system of the mouse embryo [34]. Retinoic acid can During the periattachment period, bovine stimulate a cascade of cellular remodeling steps conceptuses are dependent upon uterine secretions possibly by inducing cell production of for nutritional need, and RBP originating from the transforming growth factor-β (TGF-β), a major conceptus may serve to transport retinol from modifier of extracelluar matrix [16]. Bovine uterine tissues to the embryo. The present study trophoblast cell-conditioned medium has both demonstrated secretion of RBP by the day 13 TGF-α and TGF-β activities [35]. TGF-β has also spherical blastocyst. It appears to indicate that been shown to stimulate expression of the retinol is needed for the initiation of early epidermal growth factor receptor [36]. The present embryonic development. Elongation is believed to study demonstrated that trophoblast cells were the be mediated through gene activation within the sites of RBP synthesis. It is likely that some of conceptus [16]. Increased secretion of RBP by day actions of retinoids on embryonic cell proliferation 14–15 elongating conceptuses, observed in the and differentiation may be related to the functions present study, is coincident with the timing of of growth factors and their receptors. RBP IN BOVINE CONCEPTUS 429

The mechanisms that affect conceptus RBP the developing conceptus is coincident with rapid secretion are unclear. Our preliminary studies proliferation and differentiation of extraembryonic showed that bovine conceptuses treated with membranes, and initiation of growth of the embryo retinol (0.35 µM) secreted three time as much proper. The events of early embryonic immunoreactive RBP than conceptuses without development may be indicative of an important retinol treatment. Thus, retinol may upregulate role for conceptus RBP in the transport, availability conceptus RBP secretion in vitro. Similar and metabolism of retinol during early pregnancy. observations from rat liver and visceral yolk sac have indicated that retinol nutritional status affects RBP secretion but not biosynthesis. When retinol Acknowledgement stores are depleted, RBP synthesis remains unchanged but RBP secretion is inhibited [37, 38]. This study was supported partly by the grant Yolk sac is considered as fetal liver during early (1999 AST-2.1-AID-01(9)) from the Council of pregnancy. The liver plays a central role in retinoid Agriculture of Republic of China. The guidance metabolism and storage. The liver is the major site and assistance of Dr. James D. Godkin in of plasma RBP synthesis [39]. Whether the Department of Animal Science, University of conceptus and liver share the same mechanism for Tennessee, Knoxville, are gratefully acknowledged. controlling secretion of RBP still needs further I also wish to thank Dr. Xuixiong Goa for assistance investigation. with immunocytochemistry procedures. In summary, synthesis and secretion of RBP by

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