Translocation of the Zinc Finger Protein Basonuclin from the Mouse Germ Cell Nucleus to the Midpiece of the Spermatozoon during Spermiogenesis¹

^d Department of Dermatology and ^e Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

	ABSTRACT

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Basonuclin was first described as a human keratinocyte zinc finger protein present in the nuclei of proliferative basal keratinocytes in the epidermis. It disappears from keratinocytes that have lost their proliferative ability and have entered terminal differentiation. We now report that basonuclin is present also in the germ cells of the mouse testis and ovary. Immunocytochemical staining detected basonuclin in the nuclei of spermatogonia and spermatocytes at various developmental stages. During spermiogenesis, it relocated from the nucleus to the midpiece of the flagellum of the spermatozoa. In the ovary, basonuclin was found mainly in the nuclei of developing oocytes. The dual presence of basonuclin in differentiated spermatozoa and oocytes suggests that it may play a role in their differentiation and the early development of an embryo.

	INTRODUCTION

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Basonuclin was first described as a keratinocyte zinc finger protein with sequence motifs similar to those of other transcription factors [1]. In the epidermis, it is found exclusively in the nuclei of the basal keratinocytes. Basonuclin expression is down-regulated when keratinocytes lose their proliferative ability and commence differentiation [2]. Unlike the Ki67 antigen, a common cellular proliferative marker found exclusively in dividing cells [3, 4], basonuclin is present not only in dividing keratinocytes but also in keratinocytes that apparently are quiescent (no Ki67) but retain their proliferative ability [2]. We hypothesized that basonuclin is likely a transcription factor required for the maintenance of keratinocytes' proliferative ability.

Although the epidermis and the seminiferous tubules produce dramatically different terminally differentiated cells, they share similar tissue architecture and cytokinetics. In adult animals, both renewal epithelia are stratified with well-defined polarity. Stem cells in both tissues reside near or in contact with the basement membrane [5–7]. Division of the stem cells produces a population of rapidly proliferating transient amplifying cells, which forms a cellular flux leaving the basal layer. Superimposed on this cellular flux is a differentiation program, which transforms the progeny of the stem cell into highly specialized cells when they reach the topical or luminal side of the tissue.

Transcription regulation plays an important role in many if not all cellular differentiation processes. Each cell type employs a unique set of transcription regulators to achieve a particular developmental goal. This, however, does not exclude the fact that two cell lineages, such as somatic keratinocytes and testicular germ cells, share a number of transcription regulators. Aside from the common housekeeping transcription factors, the epidermal and seminiferous epithelia share a small but increasing number of transcription factors, e.g., mad [8] and the STAT (signal transducer and activator of transcription) family of proteins [9]. In a recent survey, we found that basonuclin mRNA and protein were abundant also in human testis (unpublished results). The presence of basonuclin in these two similar yet distinct epithelia is intriguing. Here we describe a study of basonuclin protein distribution and developmental regulation in neonatal and adult mouse testes.

	MATERIALS AND METHODS

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Materials

C57BL/6J, 129/sv, and BALB/cJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). Purified normal rabbit IgG, CM Affi-Gel Blue Gel, and Affi-Gel 10 affinity matrix were from Bio-Rad Laboratories (Richmond, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was purchased from Molecular Probes (Eugene, OR). Rat anti-GATA monoclonal antibody (N6/#sc-265) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rat immunoglobulin (#sc-2011) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Poly(A)⁺ RNAs of the human tissues were purchased from Clonetics (La Jolla, CA). Total mouse testis RNA was extracted by using RNAzol B (Tel-Test, Inc., Friendswood, TX). The first-strand cDNA was synthesized using a kit from Life Technology Inc. (Bethesda, MD). Two primers (5'-AGCTCAGATGAAGACATGCC and 5'-CTTTGAAGATGACAGATGTCTGGG) were used to amplify the human basonuclin cDNA sequence (785 base pairs, from nucleotides 1770 to 2555, coordinates according to [1]) under the following conditions: 94°C for 3 min, then 30 cycles of 94°C for 1 min, 52°C for 3 min, 72°C for 2 min; the final step was 7 min at 72°C. The ß-actin cDNA fragment (604 base pairs) was amplified with primers 5'-ATGGAGAAAATCTGGCAC and 5'-TGATGGAGTTGAAGGTAG (a kind gift of Stuart Lessin and Floyd Fox, University of Pennsylvania, Philadelphia, PA) under the following conditions: 94°C for 4 min, then 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C 1 min; the final step was a 7-min incubation at 72°C. One tenth of the basonuclin reaction and 1/25 of the ß-actin reaction were mixed and electrophoresed on a 1% agarose gel, and the DNA was stained with ethidium bromide. To amplify the mouse testis basonuclin cDNA, the following primers were used: 5'-CCGGAATTCGGGAAGATAAACCACCGTCAGTAT and 5'-CCGCTCGAGAGAGTTGAGGCACTGTTCTTTTTC. The conditions for the PCR were 94°C for 3 min, then 35 cycles of 94°C for 30 sec, 55°C for 1 min, and 72°C for 1 min; the final step was a 5-min incubation at 72°C.

RT-PCR-amplified DNA from the human tissues were separated by electrophoresis in a 1% agarose gel and transferred to a nitrocellulose membrane. The membrane was probed with a radiolabeled human basonuclin cDNA fragment (Southern analysis).

Generation of Rabbit Polyclonal Antibodies Against Mouse Basonuclin

A histidine-tagged basonuclin fusion protein was synthesized and used to raise antibodies in rabbits. Based on the sequence conservation between human and Drosophila, degenerate primers were designed to amplify a portion of exon 4 from the BALB/cJ mouse genomic DNA. The 0.9-kilobase PCR product was sequenced to confirm that it was indeed from the mouse basonuclin gene. This PCR product was cloned into an expression vector (pRSET; Invitrogen, San Diego, CA), and the fusion protein was produced in JM109 bacteria. Inclusion bodies were loaded on an SDS-PAGE gel; the band containing the full-length fusion protein was excised, dialyzed to remove SDS, homogenized, and emulsified in Freund's complete adjuvant for the initial boost (200 µg) and in Freund's incomplete adjuvant for the subsequent boosts (100 µg/boost). Rabbits were immunized by intradermal injection (Cocalico Biologicals, Inc., Reamstown, PA). The antiserum produced was named MBP-767. This antiserum does not react with proteins from bacteria carrying only the expression vector.

For immunofluorescence experiments, the MBP-767 antiserum was purified by first using a CM Affi-Gel Blue column and then using a fusion protein-conjugated Affi-Gel 10 column. The bound antibodies were eluted with 0.1 M glycine at pH 2.5 [2]. Purified normal rabbit IgG was used as negative control.

Isolation of Spermatogenic Cell Populations

Pachytene spermatocytes and round and condensing spermatids were isolated according to the published protocols [10, 11]. Briefly, adult mice testes were decapsulated, and cells were dissociated with sequential collagenase and trypsin-DNase I treatments. Cell populations were size separated by sedimentation over 2–4% BSA gradients in Krebs-Ringer bicarbonate medium. By this method, the purified populations of pachytene spermatocyte and round spermatid were more than 85% pure. However, the condensing spermatid population was only 40–50% pure, contaminated primarily with anucleated residual bodies and some round spermatids. Mature spermatozoa were isolated from the caudae epididymides. The cells were solubilized in SDS-sample buffer containing 40 mM dithiothreitol and heated to 100°C for 5 min. Protein concentrations were determined as described previously [12], and 5 µg of protein was loaded per lane for SDS-PAGE.

Immunoblot Analysis

Extracts from fractionated testis cells were resolved on 8% SDS-PAGE gels and transferred to nitrocellulose paper. Membranes were blocked with blocking solution (PBS, 0.05% Tween 20, 4% dry milk, 1% BSA, and 1% normal goat serum) for 1 h at 23 ± 1°C. Membranes were probed with MBP-767 antiserum (1:100) at 4°C overnight with shaking and then washed with three quick rinses followed by five rinses, within 30 min, with washing solution (PBS, 0.05% Tween 20). Bound antibody was detected with HRP-conjugated goat anti-rabbit IgG (1:1000 in blocking buffer) for 1 h at 23 ± 1°C and detected by chemiluminescence by ECL (Amersham, Arlington Heights, IL).

Indirect Immunofluorescence Microscopy

Mouse testes and ovaries were frozen in OCT (Miles Inc. Diagnostic Division, Elkhart, IN) and cryosectioned. Sections (4 µm) were fixed for 5 min each in methanol (-20°C) and acetone (-20°C) and then blocked for 1 h at 23 ± 1°C with 1% BSA in PBS. Incubations with affinity-purified MBP-767 (1:8 or 1:16) or GATA (1:50) were in 1% BSA in PBS at 4°C for 16 h. After washing, Texas Red-conjugated anti-rabbit IgG (1:400) or FITC-conjugated anti-rat IgG (1:80) was reacted with the sections for 1 h at 23 ± 1°C. After a 15-min wash in PBS, the sections were stained with Hoechst 33258 (1 ng/ml) for 2 min, rinsed briefly, and mounted for viewing and photography with a BX60 photo microscope (Olympus Corp., Lake Success, NY). To detect basonuclin in spermatozoa, epididymal sperm of a 2-mo-old mouse were isolated. The sperm (1 x 10⁶) were plated onto #1 coverslips in 6-well culture dishes and incubated for 30 min at 23 ± 1°C. The sperm were fixed for 5 min with methanol (-20°C), permeabilized by dipping briefly in acetone (-20°C), and then subjected to the same protocol for indirect immunofluorescence as the frozen OCT tissue sections.

	RESULTS

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Basonuclin mRNA Was Abundant in Human and Mouse Testis

By using the RT-PCR technique, we screened for the basonuclin sequence in equal amounts of RNAs from 12 human tissues, extending our previous survey [2]. From the intensity of the band of the amplified cDNA, it appeared that testis RNA contained the highest concentration of basonuclin mRNA and foreskin the second (Fig. 1A). The amplified cDNA fragments were probed in a Southern analysis with a previously cloned human basonuclin cDNA fragment [1]. The sequence amplified from testis cDNA hybridized equally well as the sequence amplified from the foreskin cDNA (Fig. 1B). The Southern analysis also revealed amplified basonuclin sequence from RNAs of thymus, spleen, mammary glands, placenta, brain, and heart. No signal was seen with uterus, kidney, bone marrow, and small intestine mRNAs, even by the Southern analysis (Fig. 1, A and B). The finding that testis contains basonuclin mRNA was verified when we sequenced an amplified cDNA fragment synthesized from mouse testis RNA. The sequence of this mouse testis cDNA (GenBank accession number: AF025301) is virtually identical to that of basonuclin cDNA obtained from mouse skin [13]. Both sequences are approximately 85% identical to the nucleotide sequence of human keratinocyte basonuclin cDNA, consistent with the evolutionary divergence of the two species. This analysis therefore confirms our identification of testis as another tissue with a high concentration of basonuclin mRNA.

View larger version (44K): [in this window] [in a new window]   FIG. 1. An RT-PCR analysis for the presence of basonuclin message in various human tissues (A) and a Southern analysis of these PCR products (B). Lanes: 1, bone marrow; 2, fetal brain; 3, heart; 4, kidney; 5, mammary gland; 6, placenta; 7, skin; 8, small intestine; 9, spleen; 10, testis; 11, thymus; and 12, uterus. All were adult tissues unless otherwise indicated.

Basonuclin Was Present in the Nuclei of Spermatogenic Cells

We had previously detected basonuclin in the nuclei of human testicular cells by an antiserum raised against a recombinant human basonuclin (unpublished results). Because human tissue is scarce, and to avoid complications that may arise with the use of anti-human basonuclin antibody on mouse tissue, we raised a rabbit anti-mouse basonuclin antiserum (MBP-767). This anti-mouse basonuclin antiserum detected a band of 120 kDa in the extract of spermatocytes (Fig. 2A) and stained a nuclear antigen in the basal keratinocytes of the neonatal mouse epidermis (Fig. 2B).

View larger version (65K): [in this window] [in a new window]   FIG. 2. Anti-mouse basonuclin antiserum (MBP-767) detects a band of 120 kDa on immunoblot of testis protein (pachytene spermatocytes) (A) and stains a nuclear antigen in basal keratinocytes of mouse epidermis (B). In A, lane 1, the blot was probed with preimmune serum; in lane 2, with immune serum. Molecular weight markers (x10-3) are shown to the left. In B, the dashed lines define the boundary of the epidermis, and the arrowhead indicates the position of the basal layer.

Immunocytochemical staining of mouse testis by affinity-purified rabbit MBP-767 serum showed that in the developing mouse testis (neonatal Day 24), a large number of cells in the seminiferous tubules contained basonuclin. Normal rabbit IgG, used as a control, did not stain this tissue. Because the basonuclin-containing cells were distributed over the entire stratified seminiferous epithelium, it appeared that primary and secondary spermatocytes and spermatids all expressed basonuclin (Fig. 3B). In general, basonuclin staining in these cells was confined to the nuclei as defined by Hoechst 33258, a DNA-staining dye (Fig. 3C). To investigate whether the Sertoli cells also express basonuclin, an anti-GATA-1 protein monoclonal antibody was used to stain testis sections along with MBP-767. Structurally, GATA-1 protein is similar to basonuclin in that it contains paired zinc fingers [14]. However, GATA-1 is strongly expressed only in the Sertoli cell lineage in the initial wave of spermatogenesis in the prepubertal mouse [15]. The anti-GATA antibody stained mainly the nuclei of cells along the basement membrane of the seminiferous tubules (Fig. 3A), a location consistent with that of the Sertoli cells [7]. Anti-GATA and anti-basonuclin antibodies seem to stain different populations of cells, suggesting that basonuclin might not be expressed in Sertoli cells (Fig. 3D).

View larger version (60K): [in this window] [in a new window]   FIG. 3. Basonuclin is expressed in the nuclei of spermatogenic cells in the seminiferous tubule. Prepubertal mouse testes (neonatal Day 24) were cryosectioned and stained simultaneously with a rat monoclonal antibody against GATA-1 protein (N6) (A) and the affinity-purified anti-mouse basonuclin serum (MBP-767) (B). The nuclei were visualized by DNA-staining dye Hoechst 33258 (C). Note that cells containing basonuclin (red) are distinct from the GATA-1-containing Sertoli cells (green), as shown by superimposition of basonuclin staining pattern over that of the GATA-1 (D). Bar = 50 µm. The peripheral region of a seminiferous tubule is enlarged in E to show simultaneously basonuclin stain (red) and GATA-1 stain (green).

Developmental Regulation of Basonuclin in Early Spermatogenesis

The time course of the emergence of each spermatogenic cell type in the prepubertal mouse has been well established [16]. Basonuclin was detected in the seminiferous tubule at neonatal Day 4, the earliest time we investigated. At this time, the seminiferous epithelium is composed of less than 2–3 layers of cells, of which a vast majority are Sertoli cells and only a small number are primitive type A spermatogonia of the germ cell lineage [16]. Corresponding to this scarcity of germ cells at this stage of testis development, and consistent with the notion that basonuclin is not expressed in the Sertoli cells, only a minority of cells were found to contain basonuclin within the nascent seminiferous tubules (Fig. 5A). This was made clear through comparison of the number of nuclei containing basonuclin and the total number of cells in the seminiferous tubules as indicated by the DNA-staining dye Hoechst 33258 (Fig. 5B). At a higher magnification (Fig. 5A, inset), basonuclin appeared clustered (or aggregated), as in the nuclei of basal keratinocytes (Fig. 2; [2]).

View larger version (153K): [in this window] [in a new window]   FIG. 5. Basonuclin expression during prepubertal testis development. Cryosectioned neonatal mouse testes were stained with the affinity-purified anti-mouse basonuclin serum (MBP-767) (red) and Hoechst 33258 (blue). A, C, and E are basonuclin stains; B, D, and F are basonuclin stains superimposed on Hoechst stains. A and B) Neonatal Day 4; C and D) neonatal Day 13; E and F) neonatal Day 24. A control staining (normal rabbit IgG) of a neonatal Day 24 seminiferous tubule is shown in the upper right inset in E. Note that in F, three different tints can be seen across the seminiferous epithelium (see text and also Fig. 6). The antiserum also stained nuclei of a small number of cells in the tunic albuginea (asterisk). Bar in A = 50 µm. The inset in A shows basonuclin nuclear staining of a neonatal Day 4 seminiferous epithelium, viewed at higher magnification. Bar = 10 µm. In C and E, lower-case letters mark seminiferous tubules with distinctive staining patterns: a, basonuclin-containing cells were peripheral, and cells near the lumen were not stained strongly; b, MBP-767 stained peripheral and central cells equally well; c, large basonuclin-containing cells fill most of the lumen space; and d, small basonuclin-containing cells near the luminal space—chromatin condensing is apparent, revealed as tighter, smaller DNA stain. Bar in lower left inset in E = 50 µm.

By neonatal Day 13, the number of spermatogenic cells (the primary spermatocytes) surpasses that of the Sertoli cells, and the number of basonuclin-containing cells also increased considerably (Fig. 5, C and D). These primary spermatocytes, the product of proliferation and differentiation of the spermatogonia, are in meiotic prophase (leptotene, zygotene, and pachytene) [16]. Several basonuclin staining patterns among adjacent seminiferous tubules were observed, corresponding to the different stages of spermatogenesis occurring at the section plane in each tubule. In one pattern, basonuclin-containing cells formed a continuous single layer and remained in the basal or near-basal location within the seminiferous tubule (Fig. 5C, a). In such tubules, cells closer to the lumen had much weaker nuclear staining, suggesting either that the protein was less accessible or that its synthesis was reduced. In the other pattern, basonuclin-containing cells with smaller nuclei, and located near the center, filled the entire tubule (Fig. 5C, b). At this stage, the most intense fluorescence was from the peripheral cells. From this time course, we concluded that basonuclin was present in both spermatogonia and primary spermatocytes.

By postnatal Day 24, round spermatids become dominant in the luminal epithelium, and two types of basonuclin-containing nuclei coexisted in adjacent seminiferous tubules. In one type, the nuclei were very large and stained brightly. They sometimes occupied the center of the tubule and encroached on the luminal space (Fig. 5E, inset, c). These were likely nuclei of pachytene spermatocytes. The other type had very small nuclei in which chromatin condensation had occurred (Fig. 5E, d, and F). These were likely round or early condensing spermatids. In contrast to the immune serum, no stain was seen when control normal rabbit IgG was used on this tissue (Fig. 5E, inset, upper right). In these tubules, when the image of basonuclin staining was superimposed on the Hoechst staining of the same field, three color variations were seen (Fig. 5F). The peripheral cells had a bluish tint of the Hoechst dye. These were likely Sertoli cells that had no basonuclin. The smaller germ cells near the lumen had an orange-red color; the larger cells closer to the basement membrane had a bluish-pink tint, due to overlap of red fluorescence (basonuclin) with the blue fluorescence (DNA) (Figs. 4 and 5F). In the smaller cells located near the lumen, the zone of basonuclin staining appeared larger than that of the DNA stain, suggesting that basonuclin was beginning to be translocated into the cytoplasm (Fig. 4, A–C). As will be described in the next section, this process leads to the complete removal of basonuclin from chromatin in the mature spermatozoa.

View larger version (0K): [in this window] [in a new window]   FIG. 4. Basonuclin is sequestered from the nucleus during spermatid chromatin condensation. Shown in A–C is the same section of the seminiferous epithelium of a 24-day-old mouse. The lumen is beyond the top of the photograph. A) Hoechst stain alone, B) basonuclin stain alone, and C) basonuclin stain superimposed on Hoechst stain. Note in cells near the basement membrane, the area defined by basonuclin stain follows closely that of the DNA stain (arrow); but in spermatids near the lumen, whose chromatin condensation is apparent, the area of basonuclin stain is larger than that of the DNA stain (arrowhead). D) An MBP-767 immunoblot of protein extracts from fractionated seminiferous cell populations. Bands revealed by immunoblot for basonuclin are indicated by arrowheads on the right. Seminiferous cell populations used in the blots: lane 1, pachytene spermatocytes; 2, round spermatids; 3, condensing spermatids. Note the increase in apparent molecular weight of basonuclin band in lane 3 as compared to lanes 1 and 2.

Translocation of Basonuclin during Spermiogenesis

In the final stage of spermatogenesis, spermiogenesis, spermatids approaching the end of their differentiation can be seen with their tails in the lumen and their heads still embedded within the epithelium. In these spermatids, basonuclin was no longer associated with the nucleus; instead, two new locations were detected. The tail, or at least a portion of the tail, was stained brightly by MBP-767 antiserum (Fig. 6B, asterisk). Also seen was a staining pattern of fine dots, which were located only in the region of luminal epithelium where basonuclin nuclear staining had disappeared (Fig. 6, A and B, arrowheads). We have not been able to identify the subcellular structure associated with this staining. Corresponding to this redistribution of the protein, we also detected on Western blots an increase of the apparent molecular weight of basonuclin in condensing spermatids, suggesting a modification of the protein (Fig. 4D). The redistribution of basonuclin during spermiogenesis was confirmed by studying basonuclin localization in mature sperm. Spermatozoa isolated from the epididymis contained basonuclin, but the most intense fluorescence was associated with the midpiece of the sperm, not the head (Fig. 6C). No DNA staining was detectable in the midpiece. In the neck of the sperm, the antibody detected two small structures (dots) that were at the connecting piece of the tail separated from the midpiece. They are likely the centrioles (Fig. 6C, ct) from which the flagellum originates. The acrosome of spermatozoa was also stained by the MBP-767, albeit weakly. Basonuclin protein was thus found in cells throughout the entire spermatogenesis process.

View larger version (94K): [in this window] [in a new window]   FIG. 6. Redistribution of basonuclin during spermiogenesis and its localization in spermatozoa. Shown in A is a cryosectioned seminiferous tubule stained with the affinity-purified anti-mouse basonuclin serum (MBP-767). The region framed in A is enlarged and shown as DNA stain (Hoechst 33258) superimposed on basonuclin stain (B). Note the nuclear staining (arrow) and the fine dotted staining outside the nucleus (arrowheads). A section of a bundle of tails was also brightly stained (asterisk). An epididymal spermatozoon was stained with MBP-767 and Hoechst (C). The midpiece of the tail was stained most intensely. Staining of centrioles (ct) was also detected (arrowhead). The head of this sperm is enlarged in the inset. Abbreviations: lu, lumen; hd, head; mp, midpiece; pp, principal piece; nk, neck; ac, acrosome; ct, centrioles. Bars for A and C = 50 µm and 10 µm, respectively.

Basonuclin in Oocytes

The presence of basonuclin in the midpiece of sperm suggested that it is carried by the sperm into the oocyte, since the entire sperm is incorporated into the oocyte cytoplasm during fertilization [17]. The fact that sperm carry basonuclin into the oocyte may imply that oocytes do not express the protein themselves. We thus examined immunocytochemically the presence of basonuclin protein in adult mouse ovary. Affinity-purified MBP-767 detected basonuclin in the nuclei of oocytes within the primary follicles, which are outlined by a single layer of granulosa cells (Fig. 7). Basonuclin was present also in secondary oocytes, which have just undergone the first of the two maturation (meiotic) divisions (not shown). We do not know whether basonuclin is expressed in the primordial follicle at this time. Weak or no staining was detected in granulosa cells of the follicle and corpus luteum and in theca cells of the ovarian cortex. The fact that in the ovary, only oocytes contained significant amount of basonuclin may explain why basonuclin mRNA cannot be detected by RT-PCR method using whole-tissue RNA [18].

View larger version (0K): [in this window] [in a new window]   FIG. 7. Basonuclin in ovary. Shown is a developing oocyte in its primary follicle from a C57BL/6J mouse. A) Hoechst stain, B) basonuclin stain, and C) the two stains superimposed. Note that the fluorescence in the nucleus of the oocyte is far more intense than that of the follicle cells. Abbreviations: O, oocyte; os, ovarian stroma; and fc, granulosa cells.

	DISCUSSION

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Since the discovery of basonuclin cDNA from keratinocytes [1], there have been several reports to indicate that the expression of basonuclin is not restricted to this cell type. This study, as well as other recent reports [18, 19], shows that basonuclin mRNA and protein are expressed in testis. Basonuclin's wider distribution in both dividing and differentiated testicular germ cells may explain why RT-PCR analysis of testis tissue RNA gave a stronger signal than that of the skin, where basonuclin is restricted to the basal cells of the epidermis (Fig. 1A). By the same token, the lower-level basonuclin message in thymus, spleen, mammary gland, and placenta may indicate that like the ovary, these tissues could contain a small number of cells that express a high level of basonuclin. The spectrum of tissues in which basonuclin is expressed is therefore broader than previously thought.

A notable discrepancy between the present study and that of Yang et al. [18] is the intracellular location of basonuclin. By using anti-basonuclin peptide antibodies, Yang et al. [18] detected an antigen associated with the centrosomes of the primary (or secondary) spermatocytes located in the middle to inner sections (i.e., near the lumen) of the germ cell epithelium in the seminiferous tubule in the adult mouse. Those antibodies also detected basonuclin in the acrosome as well as the midpiece of the sperm. Our results agree with those of Yang et al. in that basonuclin is associated with the midpiece and possibly centrioles of spermatozoa. We also detected occasional staining of the acrosome of the sperm. Our detection of basonuclin in neonatal Day 4 testis is consistent with their in situ hybridization result, which showed basonuclin message in the newborn testis. Our antiserum, however, reproducibly stains the nuclei of developing germ cells in the seminiferous tubule from neonatal to adult animals, whereas that of Yang et al. did not. One possible explanation for this discrepancy is that different staining patterns reflect different conformations of basonuclin. Since the three peptide sequences used by Yang et al. as antigens were of human origin and located in the N-terminal half of the molecule, whereas the sequence of our larger fusion protein was of mouse origin and included C-terminal sequences, it is conceivable that different staining patterns reflect varying accessibility of the antigens.

As described in the present study, basonuclin appears to localize within the nuclei of the germ cells during spermatogenesis and oogenesis, with the exception of the spermatozoa. Its nuclear localization is consistent with a role as a transcription regulator as previously proposed [1, 2]. Nuclear transcription activities are gradually shut down during the chromatin condensation in spermiogenesis, in which somatic-type histones are replaced initially by transition proteins and then by protamines, resulting in a higher degree of chromatin condensation than in any mitotic chromosomes [20–23]. Remarkably, it is precisely during late spermiogenesis that basonuclin is sequestered from the nucleus. It has been reported that phosphorylation of a serine residue downstream from the nuclear localization signal in the human basonuclin promotes cytoplasmic localization of the protein [24]. Since this serine is conserved in the mouse [13], we hypothesize that the increase in molecular weight of basonuclin in condensing spermatids may reflect modifications that include the phosphorylation of this serine, which results in basonuclin's departure from the nucleus.

Although it leaves the nucleus of the condensing spermatids, basonuclin remains in the cell and is incorporated into the mature spermatozoon. This behavior, though puzzling, is not unique. In the mouse, the transcription factor Stat4 was found recently to associate with the perinuclear theca of the spermatozoa. Stat4 is located in the nucleus as well as in the cytoplasm of the haploid spermatids [25]. Since neither the mRNA nor the protein of Stat4 was detected in oocytes, it was hypothesized that Stat4 may be a paternally contributed factor for early embryonic development. Such a concept was supported by a study of the gene for Spe-11 in Caenorhabditis elegans. Mutants of Spe-11 have a paternal-effect embryonic-lethal phenotype. It was suggested that since oocytes do not express the gene, Spe-11 is delivered by the sperm to the egg to perform vital functions in early development [26]. However, basonuclin is also abundantly expressed in maturing oocytes. Therefore, the reason for sperm to carry the protein is not obvious. But it is clear that basonuclin's localization in both sperm and oocyte would ensure its presence at the time of fertilization and suggests that this protein has a role at the beginning of life, in the earliest embryos.

	ACKNOWLEDGMENTS

 
The authors wish to thank Drs. Howard Green, Shiro Iuchi, and Kyoichi Matsuzaki for communicating unpublished results; Drs. George Murphy and John Tomaczeski for frozen sectioned testis tissues; Drs. Stuart Lessin and Floyd Fox for providing the ß-actin PCR primers; Dr. Peter J. Koch for suggestions; Drs. Howard Green, Robert Lavker, Tung-Tian Sun, and Sigrid Regauer for their critical reading of the manuscript.

	FOOTNOTES

 
¹ This work was supported by grants from University of Pennsylvania Research Foundation, University of Pennsylvania Cancer Center, NIH (AG14456) (HD33052) and USDA (95-37203-1982). M.G.M. is a recipient of a Dermatology Foundation Research Fellowship. H.T. is a recipient of a Dermatology Foundation Career Development Award. GenBank accession number: AF025301.

² Correspondence: Hung Tseng, Stellar-Chance Laboratories, M8c, 422 Curie Blvd., Philadelphia, PA 19104. FAX: (215) 573-9102; htsengpe{at}mail.med.upenn.edu

³ Current address: Pharmacology WP46-300, Merck & Co., Inc., P.O. Box 4, West Point, PA 19486-0004.

Accepted: March 25, 1998.

Received: February 5, 1998.

	REFERENCES

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