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Expression of Bone Morphogenetic Protein 7 in Murine Epididymis Is Developmentally Regulated

^a Department of Veterinary Pathobiology, University of Missouri College of Veterinary Medicine, Columbia, Missouri 65211

	ABSTRACT

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Bone morphogenetic proteins (BMPs) have been shown to play a role in the functional maintenance of the adult epididymis. To begin to investigate the role of BMP signal transduction during postnatal epididymal development, we examined the expression profile of Bmp7 in murine epididymis by in situ hybridization. Our data show that during early postnatal development (younger than 3 wk of age), Bmp7 transcripts are detected uniformly in epithelial cells throughout the epididymis. As the mice aged (from 3 to 4 wk), Bmp7 expression was gradually restricted to the initial segment, with increased levels. Bmp7 expression in the rest of the caput and corpus regions became undetectable after 4 wk of age. However, after 4 wk of age, an ascending gradient of Bmp7 expression was observed in the epididymal epithelial cells in the transition from the cauda epididymal tubule to the vas deferens. Such a unique expression profile of Bmp7 strongly suggests that epididymis-produced BMP7 may play a role in the development and functional maintenance of the epididymis, and that Bmp7 expression in the epididymis is developmentally regulated.

	INTRODUCTION

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Bone morphogenetic proteins (BMPs) constitute a family of secreted polypeptides of the transforming growth factor beta (TGFß) superfamily. Recent progress has revealed that these proteins have a wide spectrum of biological activities, from controlling cell proliferation and differentiation to regulating cell death and cell fate determination [1, 2]. On the basis of sequence identity, BMPs are grouped into several subfamilies. The decapentaplegic (Dpp) and 60A subfamilies are among the best characterized. In a given animal species, multiple Bmp-like genes are usually present in the genome. Although each member has a unique expression pattern during development and organogenesis, several genes often have overlapping expression domains and thus may have overlapping functions. For example, Bmp2 and Bmp4 of the Dpp subfamily are expressed at high levels in cardiac primordia [3–5], while Bmp8a and Bmp8b of the 60A class are expressed in stage 6 to 8 round spermatids [6–8]. Bmp7 and Bmp8a are expressed in the initial segment of the adult epididymis [8].

For the past few years, significant progress has been made in revealing the functions of various Bmp genes. In the mouse, numerous Bmp genes have been inactivated through induced and natural mutations. Unique phenotypes have been observed in most mutants. In general, mutant phenotypes are usually associated with specific expression domains or high levels of expression of a particular gene. For example, Bmp4 is expressed at high levels in the posterior and ventral mesoderm during gastrulation. Without a functional Bmp4 gene, embryos die as a result of defective mesoderm formation [9]. Inactivation of Bmp2 results in defects in heart and amnion development [10]. Mutations in the 60A class often result in defects of the urogenital system. For example, kidney defects have been observed in Bmp5 and Bmp7 mutants [11–13]. Defects in spermatogenesis have been revealed in Bmp8a and Bmp8b mutants [7, 8]. Furthermore, Bmp8a expression is detected in the initial segment of the adult epididymis. Consequently, a small proportion of the mating Bmp8a mutants show degeneration of epididymal epithelium [8].

The epididymis is a multifunctional male accessory organ [14, 15]. It provides an adequate environment for the final maturation of sperm. Therefore, the proper development and functional maintenance of the epididymis are essential for normal fertility. The epididymis develops from the Wolffian duct during embryogenesis. Its growth and functions are androgen dependent throughout development and adult life [16]. During embryonic and postnatal development, epididymal epithelial cells undergo rapid proliferation and expansion. Gross morphology shows that the highly convoluted single tubule of the adult epididymis is divided into three major segments: caput, corpus, and cauda [14]. Each region has distinct functions and expresses specific proteins. For example, the efferent duct and the caput epididymidis are responsible for the resorption of testicular fluid, and estrogen receptor plays an important role in this process [17, 18]. The c-ros tyrosine kinase receptor is required for cell fate specification of the initial segment. Inactivation of the c-ros gene results in the absence of the initial segment and male infertility despite the presence of morphologically normal sperm [19]. This clearly demonstrates that a normal, intact epididymis is essential for the functional maturation of the sperm.

Testicular-derived proteins or growth factors are also needed to maintain normal functioning of the epididymis [16]. Ligation of the efferent duct or castration with supplemental androgens results in flattening and death of epididymal epithelial cells [20, 21]. BMPs appear to be among the testis-derived factors that support the normal integrity and function of the epididymis. As reported previously, the removal of one allele of Bmp8b genes, whose expression is strictly male germ cell-specific, exacerbates the epididymal defects of the Bmp8a heterozygous mutants [8]. It is also noted that BMPs produced by testes or the initial segment of the epididymis are required for the integrity and function of the distal regions of the epididymis (distal caput and cauda) [8]. Bmp8a is expressed at low levels, while Bmp7 is expressed at high levels in the initial segment of the adult epididymis. Bmp8a mutation results in increased cell death of epididymal epithelial cells in a small portion of the mutants. Such a low phenotypic penetrance is most likely due to the production of high levels of Bmp7 proteins in the same cell types. In order to investigate the functions of BMPs in the development and functional maintenance of the epididymis, we have examined Bmp7 expression in the epididymis during postnatal development.

	MATERIALS AND METHODS

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Tissue Collection and Processing

Outbred mouse strains of ICR and 129 x Black Swiss were maintained with a 12L:12D light cycle. Mice of various ages were killed by CO₂ inhalation followed by cervical dislocation. Epididymides and testes were dissected out, rinsed in PBS, and then fixed in 4% paraformaldehyde-PBS for 2–24 h depending on the tissue sizes. After fixation, epididymides were dehydrated in a series of increasing concentrations of ethanol and stored in 100% ethanol at -20°C until further processing. Before embedding, the dehydrated epididymides were cleared in ethanol/xylenes mixture (1:1) once and xylenes twice. Embedding was accomplished after three changes of Paraplast (Fisher Scientific, Pittsburgh, PA) at 58°C (30 min each). All epididymides were sectioned at 7 µm and mounted on Superfrost Plus slides (Fisher) for in situ hybridization under RNase-free conditions.

Riboprobe Labeling

A 900-base pair murine Bmp7 cDNA fragment including part of the coding region and part of the 3' untranslated region in pGEM-7Z was used to generate antisense and sense control riboprobes [22]. Probes were labeled with [-³⁵S]UTP (Amersham, Piscataway, NJ) to a specific activity of 10⁹ dpm/µg. A final probe concentration of 2 x 10⁴ dpm/µl was used for hybridization.

In Situ Hybridization

In situ hybridization procedure was followed essentially as previously described [6–8, 23–26]. Slides were deparaffinized by two changes of xylenes followed by two changes of 100% ethanol. Rehydration was carried out in a series of decreasing concentrations of ethanol. Proteinase K digestion was performed in 50 mM Tris-HCl and 5 mM EDTA (pH 7.8) at room temperature for 8 min to facilitate probe penetration. Hybridization was carried out at 63°C for 20 h, and two high-stringent washes were performed at 65°C in 50% formamide/double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) for 30 min each. After hybridization, slides were dipped in NBT-2 emulsion (diluted with equal volume of H₂O), air dried overnight, and stored at 4°C for 7 days. Slides were developed and counterstained in Mayer's hematoxylin to reveal the nuclei of various cell types and then photographed both in dark- and brightfields for low-power magnifications. Only brightfield photographs were taken for high-power magnifications to show the localization of silver grains in different cell types. Silver grains in 10 cross-sectioned epididymal tubules in caput, corpus, and cauda regions of different ages were counted under the microscope. The mean number of silver grains per cell in each of the 10 tubules was used to obtain the mean number and standard error in each region.

	RESULTS

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Bmp7 Transcripts Were Detected in Epididymal Epithelium during Early Stages of Postnatal Development

In situ hybridization using specific riboprobes against Bmp7 mRNA revealed Bmp7 transcripts in the epididymis of newborn mice at low levels (data not shown). Similarly, Bmp7 messages were also detected at low levels in epididymal tubules at 2 days postparturition (dpp) (Fig. 1A). The levels of Bmp7 expression increased as the mice aged from 4 dpp to 2 wk (Fig. 1, B–D). Therefore, a gradual up-regulation of Bmp7 expression was observed from birth to 2 wk of age. High-power magnification photomicrographs of different regions of the epididymis further revealed that Bmp7 messages were mainly present in the epithelial cells of the epididymis (Fig. 2). The levels of Bmp7 messages increased to about 4-fold as the mice aged from 4 days to 2 wk (Table 1, based on the density of silver grains associated with the epithelial cells). Furthermore, the expression of Bmp7 in the cauda epididymidis was transiently detected in the developing smooth muscle layer adjacent to the epithelial cells (Fig. 2, C, F, and I). This transient expression peaked at 1 wk of age (Fig. 2F).

View larger version (166K): [in this window] [in a new window]   FIG. 1. Expression of Bmp7 in the epididymis during early postnatal development by in situ hybridization. Brightfield photomicrographs on left show histology; corresponding darkfield micrographs on right show hybridization signals. A) In situ hybridization of an epididymis section of a 2-dpp mouse. Hybridization signals were detected in epididymal epithelial cells at low levels. B) In situ hybridization of an epididymis section of a 4-dpp mouse. Hybridization signals are associated with epididymal tubules. C–D) In situ hybridization of epididymis sections of 1- and 2-wk-old mice, respectively. Bmp7 expression remains ubiquitous, but with increased levels. Asterisk, caput epididymidis; arrow, initial segment; arrowhead, cauda epididymidis. Bar = 120 µm in A–C, 240 µm in D.

View larger version (118K): [in this window] [in a new window]   FIG. 2. Distribution of Bmp7 message in different regions of epididymis during early postnatal development. Only brightfield photomicrographs are shown to reveal the relationship of silver grain distribution and cell types. A–C) Sections of an epididymis of a 4-day-old mouse. Bmp7 hybridization signals (silver grains) were mainly restricted to the epithelial cells. Very low levels of silver grains were also associated with the developing cells surrounding the epithelia of the cauda. D–F) Sections of an epididymis of a 1-wk-old mouse. The levels of Bmp7 expression increased as revealed by dense silver grains. Hybridization signals were restricted in the epithelial cells of caput and corpus (D and E); however, in the distal cauda region, the numbers of silver grains associated with the developing smooth muscle cells were increased (F). G–I) Sections of an epididymis of a 2-wk-old mouse. Patterns were similar to those found in (D–E), but the numbers of silver grains in the smooth muscle layer had decreased (F). Bar = 40 µm.

Bmp7 Expression Was Up-Regulated in the Initial Segment and Down-Regulated in Other Segments after Midpuberty

Before 3 wk of age, Bmp7 messages were detected with relative uniformity in the epididymal epithelial cells (Figs. 1 and 2). At 3 wk of age, the expression of Bmp7 began to increase in the initial segment and to decrease in other regions (Fig. 3A). From 4 wk to adulthood, expression of Bmp7 was detected in the initial segment at high levels and gradually decreased in the caput and corpus regions (Fig. 3, B–D). Photomicrographs of high-power magnification revealed that dense silver grains were associated with the epithelia of the initial segment (Fig. 4, A, D, and G). Figure 4, B and C, shows moderate density of silver grains in the proximal caput and cauda regions at 3 wk of age. As the mice aged, the number of silver grains decreased to background levels in the proximal caput and corpus region (Fig. 4, E, F, H, and I).

View larger version (175K): [in this window] [in a new window]   FIG. 3. Expression of Bmp7 in epididymides of midpubertal to adult mice. Brightfield and darkfield photomicrographs are on the left and right, respectively. A) A section of the initial segment and caput epididymidis of a 3-wk-old mouse. The levels of Bmp7 hybridization signals began to increase in the initial segment and decrease in other regions. B) A section of an epididymis of a 4-wk-old mouse; expression of Bmp7 in the initial segment was much higher than in other regions. C) A section of an epididymis of a 6-wk-old mouse; high levels of Bmp7 expression are restricted to the initial segment. D) A section of an epididymis of a 4-wk-old mouse, hybridized with a sense control riboprobe for Bmp7. No specific hybridization signal was observed. Arrow for initial segment, asterisk for caput epididymidis, and @ for corpus epididymidis. Bar = 240 µm in A, 480 µm in B–D.

View larger version (128K): [in this window] [in a new window]   FIG. 4. Bmp7 expression in different regions of epididymides of mid- to late-pubertal mice in high-power magnification photomicrographs. A) Initial segment of a 3-wk-old mouse. Dense silver grains are associated with epithelial cells. B) Proximal caput epididymidis of a 3-wk-old mouse. Lower levels of silver grains are associated with epithelial cells as compared to the initial segment. C) Cauda epididymidis of a 3-wk-old mouse. The density of silver grains associated with epithelial cells was similar to that for proximal caput (B). D–F) Initial segment, proximal caput, and corpus epididymidis of a 4-wk-old mouse. High levels of Bmp7 expression were detected in initial segment (D) and very low levels of Bmp7 expression in the caput (E) and corpus (F). G–I) Initial segment, proximal caput, and corpus epididymidis of a 6-wk-old mouse. High levels of Bmp7 expression were found in the initial segment (G); very low levels or no Bmp7 expression was detected in caput and corpus regions (H and I). In, initial segment; Pr, proximal caput; Ca, cauda epididymidis; Co, corpus epididymidis. Bar = 40 µm.

An Ascending Gradient of Bmp7 Expression Was Observed during the Transition of Cauda Epididymidis to Vas Deferens

From 4 wk of age to adulthood, the expression of Bmp7 was up-regulated in the initial segment of the epididymis and down-regulated in regions from the proximal caput to the end of the corpus. However, Bmp7 expression reappeared at the junction between the cauda epididymidis and the vas deferens. Figure 5 shows in situ hybridization on a section across the transition area from the cauda epididymidis to the vas deferens of a 6-wk-old mouse. The region of the epididymal tubule close to the corpus expressed Bmp7 at very low levels, while the tubule close to the vas deferens expressed Bmp7 at high levels. Tubule sections in the middle of the cauda region contained intermediate levels of expression depending upon the relative distance to the corpus and vas deferens.

View larger version (120K): [in this window] [in a new window]   FIG. 5. Bmp7 expression in cauda epididymidis of a 6-wk-old mouse. A section through the cauda epididymidis is shown on top, and darkfield revealing Bmp7 hybridization is shown at bottom. Arrowhead indicates that the region of epididymal tubule close to the corpus did not express Bmp7 at significant levels; arrow shows that the region of epididymal tubule close to the vas deferens expressed Bmp7 at high levels. Epididymal tubules between the arrowhead and the arrow expressed moderate levels of Bmp7. Bar = 240 µm.

	DISCUSSION

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The biological interactions between epididymal epithelium and spermatozoa are poorly understood. These complex processes require normal development, differentiation, and functional maintenance of epididymal structures. In order to understand the normal development and functional maintenance of the epididymis, we have to dissect the molecular mechanisms controlling these processes. Identification of specific gene expression will be the first critical step required for achieving this goal.

Numerous studies have addressed the physiological and biochemical aspects of epididymal functions [21, 23, 27]. However, the processes governing epididymal development from the newborn to puberty is an underrepresented area in the field of developmental and reproductive biology. Proper development of the epididymis requires molecular signaling pathways mediated by steroid hormones, such as estrogen and androgen, and various growth factors. Our investigation using gene expression and loss-of-function studies have implicated Bmp genes in the maintenance of normal epididymal integrity. In the absence of a functional Bmp8a gene, there is an increase in the degeneration of epididymal epithelial cells through necrosis [8]. Furthermore, the germ cell-produced BMP8B protein also has a similar role in maintaining the integrity of epididymal epithelial cells. The fact that Bmp8a is expressed in the initial segment of the epididymis at relatively low levels and only a small percentage of Bmp8a mutants show epididymis degeneration supports the notion that the expression of other Bmp genes compensates for the loss of Bmp8a. Indeed, high levels of Bmp7 expression have been detected in the same cell types as Bmp8a in the epididymis of adult mice [8]. Thus, based on the levels of expression, it is likely that Bmp7 plays a more important role than Bmp8a in the development and functional maintenance of the epididymis.

The low levels of Bmp7 expression in neonatal epididymides suggest that Bmp7 does not have a major role in early postnatal stages. However, from 4 days after birth to 3 wk of age, the levels of Bmp7 expression in the epididymis increase significantly. After 3 wk of age, regionalization of Bmp7 expression begins, and by 4 wk of age, Bmp7 expression is mostly restricted to the initial segment. The period from 4 dpp to 4 wk of age corresponds to the period of rapid cell proliferation and growth of the murine epididymis. Therefore, high levels of Bmp7 expression coincide with rapid epididymal growth, suggesting that BMP7 proteins may play important roles in cell proliferation of the epididymis. As the mice mature, the periodic expression of Bmp7 in the epididymis may reflect the physiological needs. For instance, the production and secretion of BMP7 by the initial segment alone after 4 wk of age may be sufficient to maintain the normal function for most parts of the epididymis. However, as seminal fluid passes through the epididymal tubule, the concentration of BMP7 proteins may decrease. Thus, the cauda epididymidis-produced BMP7 proteins may become necessary for maintaining the normal functions of the cauda epididymidis and/or the downstream vas deferens.

Most Bmp7 null mutants die perinatally, and a few survive to 2–3 wk after birth with severely delayed development and body size [12, 13]. Furthermore, Bmp7 heterozygous mutants do not show obvious defects in male reproduction. Therefore, the role of Bmp7 in postnatal epididymis development and functional maintenance cannot be revealed in these mutants. However, since some Bmp8a null mutants exhibit degeneration of epididymal epithelium due to insufficient BMPs, any further decrease in related BMPs, such as BMP7, will result in a more severe degeneration of epididymides. Therefore, by creating double mutants of Bmp8a/Bmp7 (Bmp8a-/-; Bmp7+/-), it will be possible to reveal whether Bmp7 plays a role in the functional maintenance of the epididymis. Experiments are under way to create such double mutants and to further examine the role of Bmp7. More sophisticated genetic approaches will have to be applied to investigate the role of Bmp7 in postnatal development of the epididymis, such as using the Cre-lox system to specifically inactivate Bmp7 in the developing epididymis.

	FOOTNOTES

 
¹ Correspondence: Guang-Quan Zhao, 209C Connaway Hall, Department of Veterinary Pathobiology, University of Missouri, College of Veterinary Medicine, Columbia, MO 65211-5300. FAX: 573 882 6827; zhaog{at}missouri.edu

Accepted: February 1, 1999.

Received: October 26, 1998.

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