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BOR - Papers in Press, published online ahead of print December 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022947
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BIOLOGY OF REPRODUCTION 70, 1080–1087 (2004)
DOI: 10.1095/biolreprod.103.022947
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Developmental Distribution of the Polyadenylation Protein CstF-64 and the Variant {tau}CstF-64 in Mouse and Rat Testis1

A. Michelle Wallace3, Toni L. Denison, Ebtesam N. Attaya, and Clinton C. MacDonald2

Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Messenger RNA polyadenylation is one of the processes that control gene expression in all eukaryotic cells and tissues. In mice, two forms of the regulatory polyadenylation protein CstF-64 are found. The gene Cstf2 on the X chromosome encodes this form, and it is expressed in all somatic tissues. The second form, {tau}CstF-64 (encoded by the autosomal gene Cstf2t), is expressed in a more limited set of tissues and cell types, largely in meiotic and postmeiotic male germ cells and, to a smaller extent, in brain. We report here that whereas CstF-64 and {tau}CstF-64 expression in rat tissues resembles their expression in mouse tissues, significant differences also are found. First, unlike in mice, in which CstF-64 was expressed in postmeiotic round and elongating spermatids, rat CstF-64 was absent in those cell types. Second, unlike in mice, {tau}CstF-64 was expressed at significant levels in rat liver. These differences in expression suggest interesting differences in X-chromosomal gene expression between these two rodent species.

gametogenesis, gene regulation, meiosis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Messenger RNA processing is one of the most important mechanisms by which diversity of gene expression is controlled in eukaryotes [1]. In our investigation of mRNA processing in male germ cells, we found that one of the most highly conserved RNA-processing elements, the canonical polyadenylation signal AAUAAA, was absent from the 3'-ends of a large number of mRNAs expressed in male germ cells [2, 3]. Furthermore, a number of significant mRNAs that were expressed in both germ cells and somatic cells used a different polyadenylation site in germ cells than in somatic cells [4–10]. This suggested key differences in the mechanism of polyadenylation in germ cells compared with that in somatic cells. In examining candidate proteins that might be involved in altering germ cell polyadenylation, we discovered that one essential polyadenylation protein, the 64 000-Mr subunit of the cleavage stimulation factor (CstF-64), had an approximately 70 000-Mr variant form that was expressed in meiotic and postmeiotic male germ cells in mice [2]. The variant protein, {tau}CstF-64 [11], also was expressed, although to a lesser extent, in mouse brain. The somatic CstF-64 protein cannot be expressed during meiosis, because the CstF-64 gene, CSTF2, is on the X chromosome, which is inactivated during male meiosis [12, 13]. This led us to the hypothesis that {tau}CstF-64 was encoded by a second gene on an autosome. The mouse gene for {tau}CstF-64, Cstf2t, was cloned and mapped to chromosome 19, confirming its existence [11], and human CSTF2T was mapped to chromosome 10 [14].

The CstF-64 is one of many X-linked genes that are inactivated during male meiosis [12]. Somatic X-chromosome inactivation in female eutherian mammals has been known for much longer [15, 16], and it likely evolved to compensate for gene dosage in the homogametic sex [17, 18]. Transcriptional silencing of sex-determining chromosomes is a phenomenon unique to mammals [19]. Unlike somatic X-chromosome inactivation that occurs in females for reasons of dosage compensation [15], X-chromosome inactivation in male eutherian mammals occurs only in germ cells. According to the leading hypothesis, because the X and Y chromosomes have only limited regions of homology, synapsis is prevented by sequestration of the X and Y chromosomes in the microscopically visible "sex vesicle" or "XY body" during the pachytene stage of spermatogenesis [12]. Interestingly, although the Xist transcript can be detected in the XY body at this time [20, 21], targeted deletion of the Xist gene, which defeats somatic X inactivation in females, has little or no effect in males [22, 23], suggesting strongly that the mechanism for male sex chromosome inactivation differs from that in females.

The CstF-64 is one of three CstF subunits required for polyadenylation. The CstF, along with poly(A) polymerase, the cleavage-specificity factor (CPSF), and two cleavage factors (CFI and CFII), can recapitulate polyadenylation in vitro [24–26]. During polyadenylation, CstF interacts with the pre-mRNA through the RNA-binding domain of CstF-64 [27] at U- or GU-rich sequences [28] within 10 to 30 nucleotides downstream of the cleavage site [29]. Interaction of CstF with CPSF promotes the cooperative binding of the 160 000-Mr subunit of CPSF to the polyadenylation signal, usually AAUAAA in somatic cells [30–32], followed by subsequent cleavage of the pre-mRNA and poly(A) addition [33–35].

We were interested to learn whether the developmental patterns of CstF-64 and {tau}CstF-64 that we saw in mice was a common pattern or one that differed among species, beginning with other rodents. We found that in rats, distribution of {tau}CstF-64 in meiotic and postmeiotic germ cells seemed to be nearly identical to that in mice, supporting its proposed role in those cells [2, 11]. Therefore, we were surprised to find that unlike in mice, the somatic CstF-64 was not expressed after meiosis in rats [36]. We were further surprised to observe that unlike in mice, rat liver expressed high levels of {tau}CstF-64. Finally, we observed that in both rats and mice, spleen and thymus express low but detectable levels of {tau}CstF-64, which is consistent with a plausible hematopoietic or immunological role for that protein. These differences between mice and rats suggest that in at least some tissues in some species, functions of the two forms of CstF-64 can substitute for one another, whereas in other tissues, the two forms might have complementary functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

In all studies, animal protocols were approved by the Institutional Animal Care and Use Committee, and NIH guidelines for animal care were followed. For developmental studies, male CD-1 mice (at 7, 13, and 17 days postpartum or retired breeders [6–12 mo of age]) and male Wistar rats (at 10, 20, and 40 days postpartum) were anesthetized with CO2 and killed by cervical dislocation. For protein analyses, tissues were removed and washed in ice-cold PBS before nuclei preparation (discussed below). Testes were quickly removed, fixed in 4% paraformaldehyde in PBS, and sectioned into 5-µm sections for histological evaluation. For studies of adult animals, male CD-1 mice (age, >35 days) and male Wistar rats (age, 62–67 days) were anesthetized with CO2, killed by cervical dislocation, and perfused by ventricular puncture with PBS followed by 4% paraformaldehyde in PBS. Testes and livers were removed and sectioned for histological evaluation.

Antibodies

The specificities of the monoclonal antibodies 3A7 and 6A9, which were raised against the human CstF-64 [37], were described previously [2]. In mice, the 3A7 antibody is specific for the somatic CstF-64, whereas 6A9 is specific for {tau}CstF-64. A control antibody, {alpha}100k-1 is a monoclonal antibody to the adenovirus 100k DNA-binding protein and is not expressed in uninfected mouse tissues.

Immunohistochemistry

Immunohistochemistry of paraformaldehyde-fixed, paraffin-embedded sections of developmental rat, adult rat, and mouse testis as well as adult rat and mouse liver was done as described previously [2, 9] using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Immunoreactive proteins were developed in 0.1 M Tris-HCl (pH 7.2) with 0.2 mg/ml of 3,3'-diaminobenzidine. Slides were counterstained using Harris hematoxylin, mounted with Permount (Fisher Scientific, Houston, TX), and viewed with an Olympus BX-60 photomicroscope (Olympus, Melville, NY).

Protein Analysis

Nuclei from rat and mouse tissues and testicular tubules were prepared as described previously [2]. Samples were sonicated briefly in loading buffer [38], boiled briefly, separated by 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes for protein immunoblot analysis [2, 39]. In an effort to adjust for differing cellular protein contents in somatic tissues, sample loading was normalized to the DNA content for each tissue using the Bio-Rad Fluorescent DNA Quantitation Kit (Bio-Rad, Hercules, CA) to control for cell number. Because stages within seminiferous tubules have differing DNA contents, tubule samples were compared to other tissues comparing glyceraldehyde phosphate dehydrogenase, and the loading was adjusted accordingly (data not shown). Protein immunoblotting was performed as described previously [2].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of CstF-64 and {tau}CstF-64 Proteins in Rat Tissues

To summarize our previous findings in mice, CstF-64 was found in the nuclei of somatic cells in every tissue examined, whereas {tau}CstF-64 was restricted to the nuclei of testicular germ cells and brain [2]. Because Cstf2, the gene for CstF-64, was found on the X chromosome, we hypothesized that the pattern of expression in mouse testis was caused by inactivation of Cstf2 during spermatogenesis and activation of the autosomal Cstf2t gene for {tau}CstF-64 [2, 11]. As further support for this hypothesis, we wished to examine CstF-64 expression in other species, including other rodents, starting with rats.

Detection of the two forms of CstF-64 was facilitated, because in mice, the 3A7 monoclonal antibody detected exclusively somatic CstF-64 whereas the 6A9 monoclonal antibody detected exclusively the variant {tau}CstF-64 [2]. To determine the distribution of the two CstF-64 isoforms in rat tissues, we used the 3A7 and 6A9 antibodies to perform protein immunoblot analysis of nuclei from several rat tissues and seminiferous tubules (Fig. 1, A and B) and compared those with tissues from mice (Fig. 1C). Protein samples from mouse testis (lane 1) and rat or mouse brain, liver, lung, spleen, testis, and thymus nuclei (lanes 2–7, respectively) were normalized to DNA content, separated by 10% SDS-PAGE, transferred to PVDF membranes, and examined by immunoblotting (see Materials and Methods). Membranes were incubated with either the 3A7 (Fig. 1A) or 6A9 (Fig. 1, B and C) monoclonal antibodies.



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  		FIG. 1. Protein blots comparing CstF-64 (A) and {tau}CstF-64 (B and C) expression in nuclei from rat (A and B) and mouse (C) tissues. Nuclei (lanes 2–5 and 7) or testicular tubules (lanes 1 and 6) were prepared as described previously [2]. Loading was normalized to nuclear DNA content as described in Materials and Methods. Samples were mouse testis (lane 1), brain (lane 2), liver (lane 3), lung (lane 4), spleen (lane 5), testis (lane 6), and thymus (lane 7)

As previously shown [2], 3A7 reacted with the approximately 64 000-Mr CstF-64 protein in mouse seminiferous tubules (Fig. 1A, lane 1). Similarly, CstF-64 was detected in nuclei prepared from rat brain, liver, lung, spleen, testis, and thymus (Fig. 1A, lanes 2–7, respectively). Slight differences in levels of CstF-64 were detected among rat tissues (Fig. 1A, compare lanes 2–7), although the significance of the differences is not clear. Also, slight differences were observed in the mobility of CstF-64 among the rat tissues (Fig. 1A, compare lanes 5–7). Because these differences do not reflect cross-reactivity of 3A7 with the more slowly migrating {tau}CstF-64 protein (data not shown; see below), differences in phosphorylation might be the cause [37, 40]. Finally, differences were observed in relative mobility between rat and mouse CstF-64 that might be caused by differential phosphorylation of the CstF-64 proteins or that might represent a species difference between the proteins (Fig. 1A, compare lanes 1 and 2).

In agreement with our earlier findings [2], the 6A9 antibody detected a protein of approximately 70 000 Mr in mouse seminiferous tubules (Fig. 1, B, lane 1, and C, lane 6). Also, in agreement with those findings, a similarly sized protein was detected in rat brain (Fig. 1B, lane 2) and testis (Fig. 1B, lane 6). These findings are consistent with the pattern seen for the {tau}CstF-64 protein in mouse (Fig. 1C, lanes 2 and 6). However, unlike what we reported earlier in the mouse [2], {tau}CstF-64 was detected in rat spleen and thymus (Fig. 1B, lanes 5 and 7, respectively).

More unexpectedly, the 6A9 antibody detected high levels of {tau}CstF-64 in rat liver (Fig. 1B, lane 3), although very little was detected in mouse liver (Fig. 1C, lane 3). Similar high levels of {tau}CstF-64 were detected in liver extracts prepared from several different rats from several different preparations, whereas little or none has ever been detected in mouse liver extracts from three different strains of mouse (Fig. 1C, lane 3, and data not shown).

CstF-64 Was Detected from Earliest Times (Day 7) in Immature Mouse Testis, But {tau}CstF-64 Was Not Detected until Day 13, When Pachytene Spermatocytes Appeared in Mice

Earlier, we examined CstF-64 and {tau}CstF-64 expression in testes of adult mice [2]. To confirm and extend these results, we examined CstF-64 and {tau}CstF-64 in the testes of mice at different ages after birth (Fig. 2), during which time germ cells are developing and specific known cell types are present [41]. At 7 days after birth, the 3A7 antibody (which detects the somatic CstF-64 in mice) detected immunoreactive protein staining in nuclei of nearly every cell type, including primitive type A spermatogonia and Sertoli cells (Fig. 2A). At Day 13, 3A7 staining continued in type A and B spermatogonia and in preleptotene, leptotene, and zygotene spermatocytes (Fig. 2C). However, staining was diminished in early to midpachytene spermatocytes. This is consistent with our earlier finding that CstF-64 protein was not expressed in stage V pachytene spermatocytes [2]. Similar patterns were seen at Day 17 (Fig. 2E), at which time spermatogonia and early spermatocytes are stained but pachytene spermatocytes become more abundant and are not stained [41]. Finally, in agreement with our earlier study [2], sections from adult mouse testis show 3A7 staining in resident somatic cells (Leydig, macrophage, and Sertoli), spermatogonia, and early spermatocytes as well as round and early elongating spermatids (Fig. 2G).



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  		FIG. 2. CstF-64 and {tau}CstF-64 expression during mouse spermatogonial development. Male mice were killed at 7 days (A and B), 13 days (C and D), 17 days (E and F), or greater than 6 mo ("adult"; G and H) after birth, and testes were removed and prepared for immunohistochemistry (see Materials and Methods). Paraffin sections of paraformaldehyde-fixed tissue were incubated with either the 3A7 (CstF-64; A, C, E, and G) or 6A9 ({tau}CstF-64; B, D, F, and H) monoclonal antibodies, and antibody interactions were detected with 3,3'-diaminobenzidine (DAB; leaves a brown staining pattern). After antibody detection, sections were counterstained with Harris hematoxylin (blue). Sections incubated with an irrelevant control monoclonal antibody showed little or no antibody staining (not shown). For a discussion of predominant cell types at each stage, see Results. Magnification x40

At Day 7 (Fig. 2B), few, if any, cells were stained with the 6A9 antibody, suggesting that {tau}CstF-64 was not expressed in spermatogonia, which are developing at this time [41]. However, by Day 13 (Fig. 2D), although not earlier, a number of centrally located cells showed nuclear staining with 6A9, suggesting {tau}CstF-64 expression in pachytene spermatocytes. This result was further seen in developing pachytene spermatocytes at Day 17 (Fig. 2F). Results with the 6A9 antibody at Days 9 and 11 postpartum were consistent with these observations (data not shown). In adult mice, {tau}CstF-64 was detected in stage V pachytene spermatocytes continuously through stage XI elongating spermatids (Fig. 2H and data not shown), exactly as described earlier [2]. (These data are summarized in Fig. 6.)



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  		FIG. 6. Comparison of CstF-64 and {tau}CstF-64 protein expression during mouse and rat spermatogenesis. Top) Timeline of events during mouse (above line, 34 days total) and rat (below line, 48 days total) spermatogenesis. Middle) Expression patterns of CstF-64 (dark gray) and {tau}CstF-64 (light gray) in mouse germ cells. Bottom) Expression patterns of CstF-64 (dark gray) and {tau}CstF-64 (light gray) in rat germ cells

Unlike in Mice, CstF-64 Was Not Expressed in Meiotic or Postmeiotic Cells in Rat Testis

To examine possible differences in rodent species, we examined CstF-64 and {tau}CstF-64 in rat testicular sections using the 3A7 and 6A9 antibodies. The 3A7 antibody detected CstF-64 in nuclei of testicular somatic cells, primarily Leydig, Sertoli, and macrophage cells (Fig. 3, A–C), which is consistent with our previous findings in mice [2]. In the seminiferous epithelium, different rat germ cell types were characterized by their association with each other and with types found in different cross-sections and then categorized into 14 stages [42]. The 3A7 detected strong immunoreactive protein in spermatogonia and in leptotene and zygotene spermatocytes (Fig. 3, A–C). The intensity of 3A7 staining diminished in pachytene spermatocytes in stage II–III tubules and was undetectable in any other meiotic or postmeiotic cell types. This was in sharp contrast to 3A7 staining in mice, which was not detected in pachytene spermatocytes but which resumed in round spermatids and early elongating spermatids (Fig. 2G) [2]. (These data are also summarized in Fig. 6.)



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  		FIG. 3. Immunohistochemical localization of CstF-64 and {tau}CstF-64 expression in adult rat testis sections. Adult male rats (age, 62–67 days) were killed and perfused with paraformaldehyde, and testes were removed for immunohistological sectioning and examination. Testes were incubated with either the 3A7 (CstF-64; A–C) or 6A9 ({tau}CstF-64; D–F) monoclonal antibodies, and antibody interactions were detected with 3,3'-diaminobenzidine (DAB). Sections in A, B, D, E, and G were counterstained as for Figure 2; Sections in C, F, and H did not receive counterstain. Control sections (G and H) were incubated with {alpha}100k-1, an irrelevant antibody that does not react with mouse tissues. Roman numerals refer to the stage of the seminiferous epithelium. ES, Elongating spermatid; LC, Leydig cell; SC, Sertoli cell; SPC, spermatocyte. Magnification x40 (A, C, D, and F and x100 (B, E, G, and H)

{tau}CstF-64 Was Detected in Pachytene Spermatocytes and in Round and Early Elongating Spermatids in Rats

Using the 6A9 antibody, {tau}CstF-64 protein was not detected in resident somatic cells in adult rat testis, nor was it detected in spermatogonia (Fig. 3, D–F). The {tau}CstF-64 was first detected in stage III–IV pachytene spermatocytes and increased in intensity through stage XIV. Similarly, {tau}CstF-64 was detected strongly in round spermatids and diminished in early elongating spermatids (stage X). In these stages of spermatogenesis, the somatic CstF-64 is absent; this is very similar to the pattern seen previously in mouse testis (Fig. 2) [2]. However, unlike in mice, no cell type in rat germ cells expresses CstF-64 and {tau}CstF-64 simultaneously (data summarized in Fig. 6). No signal was detected in histochemical sections using a control antibody (Fig. 3, G and H).

{tau}CstF-64 Was First Detected at Day 20 after Birth in Rat Testis

To determine more accurately when CstF-64 and {tau}CstF-64 expression occurred, we examined their expression in testis sections from rats at different days after birth. As expected, even at the earliest time point (Day 10), the 3A7 antibody detected CstF-64 protein in somatic cell types and early germ cells, likely type A spermatogonia (Fig. 4A). At Day 20, spermatogonia were stained as at Day 10, but staining diminished with the appearance of later spermatocyte cell types (Fig. 4C). The CstF-64 was not detected in round spermatids that appeared at Day 40 (Fig. 4E).



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  		FIG. 4. CstF-64 and {tau}CstF-64 expression during rat spermatogonial development. Male rats were killed at 10 days (A and B), 20 days (C and D), 40 days (E and F), and 65 days ("adult"; G and H) after birth, and testes were removed and prepared for immunohistochemistry (see Materials and Methods). Paraffin sections of paraformaldehyde-fixed tissue were incubated with either the 3A7 (CstF-64; A, C, E, and G) or 6A9 ({tau}CstF-64; B, D, F, and H) monoclonal antibodies as for Figure 2. Sections incubated with an irrelevant control monoclonal antibody showed little or no antibody staining (see Fig. 3). For a discussion of predominant cell types at each stage, see Results. Magnification x40

In contrast, 6A9 did not detect protein in somatic cells at Day 10 (Fig. 4B), suggesting that {tau}CstF-64 was absent at those times. However, light staining was seen in cells at the periphery of seminiferous tubules (Fig. 4B, arrows); similar staining was not seen in control experiments (data not shown, but see Fig. 3, G and H). This finding suggests a window of {tau}CstF-64 expression early during rat spermatogenesis in type A spermatogonia, which is surprising, because no staining of the corresponding cell types was seen in adult rat testis (Figs. 3, D–F, and 4H) or in mice (Fig. 2, B and H).

Strong staining by 6A9 was seen in spermatocytes by Day 20 (Fig. 4D) and in round spermatids at Day 40 (Fig. 4F). This is consistent with the meiotic and postmeiotic distribution of {tau}CstF-64 seen in adult rat testis (Figs. 3, D–E, and 4H).

{tau}CstF-64 Was Detected in Hepatocytes But Not in Other Cell Types in Rat Liver

Because we detected {tau}CstF-64 protein in rat liver extracts but not in mouse liver, we wished to determine in which hepatic cell types the CstF-64 and {tau}CstF-64 proteins were expressed. Immunohistochemical analysis was performed on paraformaldehyde-fixed mouse and rat liver sections using the 3A7 and 6A9 antibodies. Using the 3A7 antibody, we confirmed that the somatic CstF-64 was expressed in the nuclei of almost every hepatic cell type in both rat (Fig. 5A) and mouse (Fig. 5F) liver, including hepatocytes and endothelial cells. In both rat and mouse, some sinusoidal endothelial cells are less highly stained using this antibody (Fig. 5A, indicated as "S"). Also, dark spots of staining seen in mouse sinusoids probably were erythrocytic staining caused by endogenous peroxidase in erythrocytes, because they are seen in 3A7-stained (Fig. 5F) and 6A9-stained (Fig. 5G) sections as well as in controls (not shown). Rat sections (Fig. 5, A–E) lacked this pattern, because the animals were more extensively perfused before fixation.



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  		FIG. 5. Expression of CstF-64 and {tau}CstF-64 in rat and mouse liver. Paraformaldehyde-fixed histological sections from rat (A–E) or mouse (F and G) liver were incubated with either the 3A7 (CstF-64; A, B, and F), 6A9 ({tau}CstF-64; C, D, and G), or a control antibody ({alpha}100k-1; E) as for Figure 2. Some sections (A and C) were incubated with hematoxylin for contrast. BD, Bile duct; CV, central vein; H, hepatocyte; HA, hepatic artery; K, Kuppfer cell; PV, portal vein; S, sinusoidal endothelial cell. Magnification x100

Using the 6A9 antibody, little or no {tau}CstF-64 was detected in any cell type in mouse liver (Fig. 5G), although peroxidase staining of erythrocytes was apparent (blue color in the majority of mouse liver nuclei was caused by the hematoxylin-and-eosin counterstain). In contrast, the 6A9 antibody detected {tau}CstF-64 in the nuclei of the majority of hepatocytes in rat liver (Fig. 5, C and D). The {tau}CstF-64 was not detected in sinusoidal endothelial cells or in endothelial cells lining the portal vein in rats. Irrelevant control antibodies showed little or no staining in either mouse (not shown) or rat (Fig. 5E) liver, although erythrocytes accumulated stain because of peroxidase activity in mouse (Fig. 5, F and G).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We undertook this study to increase our understanding of the expression of the two isoforms of the CstF-64 polyadenylation protein in rodent testes. The gene for the somatic CstF-64 protein, Cstf2, is on the X chromosome [2] and, therefore, is inactivated during the pachytene stage of spermatogenesis. This suggested the necessity for a second, autosomal isoform of CstF-64 that would function during spermatogenesis. Our previous studies identified a variant isoform of CstF-64 that we called {tau}CstF-64, the gene for which is encoded by Cstf2t on mouse chromosome 19 [11] and by CSTF2T on human chromosome 10 [14].

To complement the description of CstF-64 and {tau}CstF-64 expression in mouse somatic and testicular cells, we examined expression of both proteins in rat tissues. Whereas the overall patterns of expression of CstF-64 and {tau}CstF-64 were very similar in both rats and mice, we found a number of striking differences between the patterns of expression of CstF-64 and {tau}CstF-64 in mice and in rats (summarized in Fig. 6). Like in mice, CstF-64 protein in rats was expressed in most, if not all, somatic cell types (Fig. 1A), and {tau}CstF-64 was expressed in rat testis and brain (Fig. 1B). However, unlike in mice, {tau}CstF-64 was expressed at high levels in rat liver (Figs. 1B, lane 3, and 5, C and D), and the somatic CstF-64 was not expressed in postmeiotic male germ cells. Therefore, the major differences between rats and mice are the strong expression of {tau}CstF-64 in rat liver and the lack of expression of CstF-64 in postmeiotic rat germ cells. Because of this lack of postmeiotic expression of CstF-64, at no time during rat spermatogenesis are CstF-64 and {tau}CstF-64 both expressed.

It is not yet clear to us why CstF-64 should not be expressed in postmeiotic rat germ cells when it is expressed in postmeiotic mouse germ cells, but we suspect the reason is linked to the unusual dynamics of X-chromosome expression in male germ cells. The CstF-64 is among the X-linked genes that are inactivated during pachytene of meiosis in spermatocytes [2]. Most X-linked genes that are inactivated during male meiosis remain inactive during subsequent spermiogenesis [19, 43–45]. It is more rare that genes become active postmeiotically [46, 47], although specific examples do exist, such as Akap4, Ube-1, and Smage [48–51]. In mice, CstF-64 falls into the latter category and is expressed postmeiotically as well as premeiotically [2]. In fact, in mice, CstF-64 mRNA is greatly overexpressed after meiosis [36]. This leads to questions about the postmeiotic function of CstF-64 in mouse germ cells and whether it is dispensable in rats. Furthermore, the differences in postmeiotic CstF-64 expression in mice and rats suggest differences in X-chromosomal dynamics during and after the period when it is inactive. In our attempts to examine these differences between mice and rats, we have undertaken xenotransplantation experiments to transfer rat and mouse germ cells and to examine premeiotic and postmeiotic expression of CstF-64 (unpublished results). Our preliminary results are consistent with a model in which the postmeiotic activation of CstF-64 is a function intrinsic to the species from which the germ cells are derived and not dependent on external cues. Other experiments (unpublished results) reveal a surprising amount of evolutionary divergence of CstF-64 and {tau}CstF-64 among closely related (Muridae) and more distantly related (Sciuromorphic and Caviomorphic) rodent species. This suggests that CstF-64 function might have species-specific functions that we have not yet uncovered.

Even more perplexing are the differences in {tau}CstF-64 expression in mouse and rat liver (Figs. 1, B and C and 5). Although many metabolic differences exist between mouse and rat liver metabolism, it is hard to see any connection between those metabolic processes and nuclear mRNA processing. However, we have seen {tau}CstF-64 mRNA expressed at high levels in many mouse tissues, including liver, in which protein expression was low or absent (unpublished results). This suggests that {tau}CstF-64 expression is regulated, at least partially, at the translational level. Perhaps in rat liver the translational repression is released, whereas in mouse liver it is not.

We detected intermediate amounts of {tau}CstF-64 in rat spleen and thymus (Fig. 1B, lanes 5 and 7). Although not apparent in Figure 1C, on longer exposures we also have detected small amounts of {tau}CstF-64 in mouse spleen and thymus (not shown). This suggests a potential role for {tau}CstF-64 in cells of the immune system. For instance, changes in the site of polyadenylation in immunoglobulin (Ig) heavy-chain mRNAs during B-cell development change IgG from a membrane-bound to a secreted form. Increases in the levels of CstF-64 are sufficient to affect this change [52, 53] but might not be necessary [40, 54]. Perhaps {tau}CstF-64 is involved in this change as well.

These findings raise many questions regarding polyadenylation function, X-chromosomal gene expression, and developmental expression of germ cell-specific proteins. To answer some of these questions, we have undertaken to clone and sequence CstF-64 and {tau}CstF-64 from a large number of mammalian and nonmammalian species [14; unpublished results]. We are finding that whereas CstF-64 is conserved among all species, {tau}CstF-64 is far less constrained. The experiments described in the present study suggest that developmental expression of these two proteins also is highly variable, even between closely related rodent species. These differences and others may be the keys to uncovering answers to questions about the role of the CstF-64 gene family in tissue-specific control of gene expression.


   ACKNOWLEDGMENTS
 
The authors wish to acknowledge Dr. Shafik Khan for samples of rat testis, James Hutson and Stuart Ravnik for help in data analysis, Brandt Schneider for use of his fluorometer, and Wyatt McMahon for critical reading of the manuscript.


   FOOTNOTES
 
1 Supported by the NIH (1 R01 HD37109-01A1), the South Plains Foundation, and Houston Educational Institute. Back

2 Correspondence: Clinton C. MacDonald, Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430. FAX: 806 743 2990; clint.macdonald{at}ttuhsc.edu Back

3 Current address: Lexicon Genetics, Inc., The Woodlands, TX 77381 Back

Received: 4 September 2003.

First decision: 25 September 2003.

Accepted: 1 December 2003.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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