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Genes to Cells (2006) 11, 71-81. doi:10.1111/j.1365-2443.2005.00915.x
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Developmental expression patterns of testicular olfactory receptor genes during mouse spermatogenesis

Nanaho Fukuda and Kazushige Touhara*

Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Conclusions
 Experimental procedures
 References
 
A subset of olfactory receptors (ORs) is expressed in mammalian male germ cells. Recent studies on human and mouse sperm have suggested that calcium signaling via a testicular OR regulates sperm flagellar motility. However, it remains to be determined at what stages testicular ORs are expressed during spermatogenesis and whether each germ cell expresses one or multiple ORs. Here we examined the developmental expression profiles of several mouse testicular OR genes using an in situ hybridization technique at the cellular level. We found that OR transcripts in the spermatogenic cells are expressed in three developmental stages: late pachyten spermatocytes, early round spermatids, or late round spermatids. The OR mRNAs were condensed in a single dot-like structure within the nuclei of a subpopulation of spermatogenic cells. Double-fluorescent in situ hybridization revealed that some cells contained two dot-like signals derived from transcripts of two different ORs, suggesting that single spermatogenic cells could express more than one OR. One cell-multiple OR gene expression combined with variability in expression appears to result in heterogeneity in the repertoire of ORs expressed by individual spermatogenic cells. Although the functional consequence of heterogeneous OR expression awaits development of a methodology for characterizing OR proteins, our observations give insights into OR gene expression as well as OR function(s) in spermatogenic cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Conclusions
 Experimental procedures
 References
 
In mammals, the olfactory receptor (OR) family comprises approximately 1000 multigenes (Godfrey et al. 2004; Mombaerts 2004; Zhang et al. 2004a). These ORs are expressed by olfactory sensory neurons (OSNs) where they act as chemosensors for thousands of volatile odorous compounds (Zhao et al. 1998; Touhara et al. 1999; Wetzel et al. 1999; Kajiya et al. 2001; Katada et al. 2005). OSNs have acquired the ability to express only one OR from a single allele (Chess et al. 1994; Ressler et al. 1994; Vassar et al. 1994; Ishii et al. 2001). Positive selection at the transcriptional level of a single OR from an OR cluster and subsequent negative feedback via the translated OR proteins appears to ensure a ‘one cell–one OR’ rule in OSNs (Serizawa et al. 2003; Lewcock & Reed 2004). As a result, axons of OSNs expressing the same OR are guided to the appropriate glomeruli in the olfactory bulb (Mombaerts et al. 1996; Wang et al. 1998; Feinstein & Mombaerts 2004). This produces a precise topographic neuronal map that provides the basis for the remarkable ability of the olfactory system to discriminate thousands of odorants using a repertoire of approximately 1000 ORs (Firestein 2001; Touhara 2002; Mombaerts 2004).

A subset of the OR gene family is also expressed in mammalian male germ cells (Parmentier et al. 1992; Vanderhaeghen et al. 1993, 1997; Asai et al. 1996). Recent studies of human and mouse sperm have suggested that ORs may play a role in sperm chemotaxis (Spehr et al. 2003, 2004; Fukuda et al. 2004). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that approximately 5–10% of the ORs (~50 of ~1000 OR genes in rodents) are transcribed in testis (Vanderhaeghen et al. 1997). Further, a recent study using an OR microarray demonstrated that 66 ORs are expressed in mouse testis (Zhang et al. 2004b). The expression patterns of these testicular ORs at the protein level have not been determined because of difficulties in producing high-quality anti-OR antibodies. In situ hybridization, which examines the cellular expression of mRNA, can be used as an alternative technique for these studies. For example, using in situ hybridization, we have recently shown that mouse OR23 (MOR23) gene transcripts are stage-specifically expressed in round spermatids and that MOR23 functions as a chemoreceptor in germ cells (Fukuda et al. 2004). These observations prompted us to ask whether other testicular OR genes are also expressed in the same developmental stages and whether each spermatogenic cell expressed one or multiple ORs.

In the present study, we cloned several OR genes from mouse testis, and determined their developmental expression profiles during spermatogenesis at the cellular level. We revealed unique features of OR gene expression in spermatogenic cells: stage-specific expression during spermatogenesis, and subcellular localization of OR transcripts within the nuclei of the spermatogenic cells. We also demonstrate that single spermatogenic cells express more than one OR by using a double-fluorescent in situ hybridization technique, suggesting that the transcriptional regulation of ORs is different in the testis than in the olfactory system.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Conclusions
 Experimental procedures
 References
 
Cloning and expression of testicular OR genes

To identify the ORs expressed in testis, we performed RT-PCR using mouse testis RNA. Degenerate primers were designed from the conserved amino acid motifs of the approximately 1200–1300 ORs so that the maximum number (672) of OR genes could be theoretically amplified. Using these degenerate primers, 17 OR genes were amplified from the mouse testis cDNA. Expression of eight of 17 ORs was confirmed by RT-PCR using specific primers (MOR23, MOR31-2, MOR171-31, MOR244-3, MOR256-25, MOR144-1, MOR171-3 and MOR13-4). We also performed RT-PCR experiments using specific primers for 21 selected ORs (see Experimental procedures), which included TPCRs, a family of ORs that have been previously reported to be expressed in the mouse testis (Vanderhaeghen et al. 1997). We found that seven of 21 OR genes were amplified (MOR139-3, MOR248-11, MOR281-1, MOR127-2, MOR13-6, MOR174-6, MOR264-10). Thus, RT-PCR using testis RNA resulted in amplification of 15 OR genes.

Even though about a half of the entire OR gene family could be potentially amplified by the degenerate primers, this number (15 OR genes) was still smaller than that obtained by the microarray analysis in Zhang et al. (2004b) (66 OR genes). We therefore performed RT-PCR to examine the expression of testicular OR genes from the microarray results. As a result, one of ten selected OR genes reported by Zhang et al. (2004b) was clearly amplified from mouse testis cDNA in the condition that the OR genes examined in our study were amplified from testis (Supplementary Fig. S1). All of the ten OR genes were found to be expressed in the OE. These results suggest that the number of OR genes expressed at a significant level is smaller than that predicted by the microarray results.

Six of these 15 ORs (MOR23, MOR31-2, MOR171-31, MOR244-3, MOR139-3 and MOR248-11) were detected by in situ hybridization using the tyramide signal amplification (TSA) system (Fig. 1). These consistently included MOR23, which has been reported to be functionally expressed in mouse testis (Fukuda et al. 2004). No signal was observed with sense probes for these ORs (Fig. 1). OR transcripts were detected without the TSA system in the OE, while the TSA system was required for detection of most of the OR gene transcripts in testis by in situ hybridization using the same OR probes (Fig. 2 and Supplementary Fig. S2). The signals of MOR244-3 and MOR248-11 were detected without the amplification system, whereas MOR23, MOR31-2, MOR171-31 and MOR139-3 required the amplification system for detection. The other nine OR genes amplified by RT-PCR were not detected in testis by in situ hybridization even with the use of the signal amplification system (i.e. MOR264-10 in Fig. 2), but the signals were clearly observed in OSNs (Supplementary Fig. S2). It is possible that these nine ORs are expressed at a very low level under the detection limit of in situ hybridization experiments. These results suggest that the levels of expression in testis appear to be different for various ORs and lower than that in OSNs.



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  		Figure 1  Testicular OR genes detected by both RT-PCR and in situ hybridization. Chr. indicates the chromosomal location based on the NCBI database or UCSC genome database. RT-PCR for each OR was performed for total RNA of testis or of the olfactory epithelium (OE) with (+) or without (–) reverse transcriptase. In situ hybridization (ISH) shows representative images of a part of the seminiferous tubule containing OR mRNA signals (brown dots). No signal was observed with a MOR23 sense probe (the bottom picture). Sections were conter-stained with methyl green. ‘Zone’ indicates the area of the OR gene expression within the OE as defined by Ressler et al. (1993) In situ hybridization results for these six ORs in the OE are shown in Supplementary Fig. S2.

 


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  		Figure 2  Comparison of the expression levels of ORs between in the olfactory epithelium (OE) and in testis. Sections of the OE and testis were hybridized to DIG-labeled OR probes and signals were detected with or without the TSA system (± TSA). OR signals were observed without TSA in the OE. The signals of MOR244-3 in testis were clearly detected without TSA, while MOR171-31 transcripts were detected only when the TSA system was applied. MOR264-10 transcripts were not detected by in situ hybridization even with the use of the TSA system. Sections of the testis for the TSA system were counterstained with methyl green. RT-PCR analysis shows that MOR264-10, MOR171-31, and MOR244-3 are expressed in both testis and the OE. RT+/–, with or without reverse transcriptase. OMP, olfactory marker protein. Bar, 50 µM.

 
The six OR genes that were detected in testis by in situ hybridization appear to be members of several subfamilies of both class I and class II ORs, and they were distributed on various chromosomes (Fig. 1). Phylogenetic analysis suggests that there is no unique characteristic sequence similarity among the testicular OR genes (data not shown). They were also expressed in a variety of receptor zones in the OE (Fig. 1 and Supplementary Fig. S2), and we did not identify an OR that was expressed only in the testis.

Developmental expression patterns of OR gene transcripts in testis

Hybridized signals were detected using anti-sense probes for testicular ORs in a subset (30–40%) of seminiferous tubules, suggesting that the ORs were not expressed during all stages of development (Figs 2 and 3A). No signal was observed with sense probes for testicular ORs (Figs 1 and 3A). The levels of expression were significantly different for various ORs (Figs 2 and 3). The transcripts for the OR genes appeared to be localized within spermatogenic cells and concentrated in a dot-like pattern (Fig. 3). This dot-like pattern was also observed without using the TSA system (Fig. 3C), excluding the possibility that the signals were an artifact of the amplification system.



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  		Figure 3  Stage-specific expression of OR genes in testis. (A) In situ hybridization in serial sections using anti-sense or sense probes for H1t, protamine 1, and three ORs. Brown signals were observed in a subset of the seminiferous tubules (asterisks). No signal was observed with OR sense probes (MOR244-3 S). (B) Stages of individual tubules in Figure 2A (numbered in the picture of MOR244-3 S) were categorized by the expression of protamine 1 (Prm1) and H1t, and by the presence of the elongated spermatids with condensed nuclei (elongate). The stages of OR expression are summarized on a map of the developmental cycle of mouse spermatogenic cells in seminiferous tubules (modified from Russell 1990). Testicular ORs were categorized into three groups according to their expression patterns. (C) A representative picture of MOR244-3 signals detected without the TSA system. The dot-like pattern was observed without amplification. (D) Double-in situ hybridization of MOR244-3 (magenta) and MOR248-11 (green) or MOR171-31 (blue). MOR171-31 was expressed in the tubules expressing MOR244-3, whereas the stages of expression for MOR244-3 and MOR248-11 were different. Bars, 100 µM.

 
The tubule-specific expression of ORs indicates that testicular ORs are expressed stage-specifically during spermatogenesis. This is consistent with our previous investigations on MOR23 (Fukuda et al. 2004). In situ hybridization analysis of serial sections revealed that different ORs are expressed in different seminiferous tubules, and thus, in different stages of development. Transcripts for MOR171-31 were found in the MOR244-3 expressing seminiferous tubules (Fig. 3A), although there was a significant difference between their levels of expression (Fig. 2). In contrast, MOR248-11 transcripts were observed in the tubules that did not express MOR244-3 (Fig. 3A).

We determined the developmental stages of each tubule by morphological examination as described by Russell (1990) and according to the patterns of protamine 1 and H1t expression in serial sections. Protamine 1 is expressed in spermatids of stages IX–XII and I–III (Mali et al. 1989) and H1t is in pachytene spermatocytes of stages V–XI (Drabent et al. 1996) (Fig. 3A,B). Seminiferous tubules can be categorized into five groups according to the expression patterns of Protamine 1 and H1t, as well as the presence of nuclei-condensed elongated spermatids (Fig. 3B). This analysis showed that the ORs are expressed in three patterns related to the mouse germ cell developmental cycle: type A, expression in late round spermatids; type B, expression in early round spermatids; type C, expression in late pachyten spermatocytes (Fig. 3B). Double-fluorescent in situ hybridization confirmed that MOR244-3 and MOR171-31 were expressed in the same seminiferous tubules, whereas MOR244-3 and MOR248-11 were expressed at different stages (Fig. 3D). Importantly, when signals for MOR244-3 and MOR171-31 were present in the same tubule, the fluorescent dot-like signals never overlapped (Fig. 3D).

Subcellular localization of OR transcripts in spermatogenic cells

To examine the subcellular localization of OR mRNAs in spermatogenic cells, we performed fluorescent in situ hybridization using anti-sense probes and counter-stained the nuclei with DAPI (4',6-Diamidino-2-phenylindole). Dot-like signals for MOR248-11 mRNA were observed within the nuclei of the pachyten spermatocytes, whereas the signals for H1t mRNA were observed uniformly in the cytosol (Fig. 4A,B). Similarly, the dot-like signals observed with MOR244-3 anti-sense probes were observed within the nuclei but not within the nucleoli of round spermatids (Fig. 4C,D). The signals for other testicular OR mRNAs were also localized within the nuclei, suggesting that OR mRNAs accumulated and condensed after transcription. No signal was obtained with OR sense probes, excluding the possibility that the signals were derived from genomic DNA (data not shown). Similar dot-like signals were observed with protamine 1 anti-sense probes in the seminiferous tubules during stages VII-VIII (Fig. 3E,F), but the dot-like signals of protamine 1 were localized in the cytoplasm of the spermatids near the nuclei. It has been reported that the protamine 1 transcripts are stored for up to 7 days after some post-transcriptional regulation before translation (Braun 1990; Dadoune et al. 2004). Thus, the unique localization pattern for OR mRNAs in nuclei suggests involvement of a previously uncharacterized mechanism for storing OR mRNA during spermatogenesis.



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  		Figure 4  Localization of OR gene transcripts in spermatogenic cells. In situ hybridization signals of OR anti-sense probes were detected using the TSA-fluorescein system, and nuclei were counter-stained with DAPI (blue). (A, B) Double-fluorescent in situ hybridization using MOR248-11 and H1t anti-sense probes. The dot-like signals for MOR248-11 mRNA (sky blue because of merging green and blue colors) were localized within the nuclei of the pachyten spermatocytes, whereas the signals for H1t mRNA (red) were observed in the cytosol. (C, D) In situ hybridization signals for the MOR244-3 anti-sense probe. Dot-like signals were observed within the nuclei but not in the nucleoli of round spermatids. It should be noted that not all of the round spermatids contained MOR244-3 mRNA signals. (E, F) In situ hybridization signals for a protamine 1 anti-sense probe. Dot-like signals were observed in round spermatid layer during stages VII–VIII. These signals were localized in the cytosolic compartment near the nuclei of the spermatid as shown in a high magnification view (F) of the dotted square area in (E). Bar, 10 µM.

 
To examine how many dot-like mRNA signals derived from a single OR were found in each cell, small pieces of seminiferous tubules were treated with trypsin after which spermatogenic cells were isolated on a slide glass, fixed and hybridized to an OR anti-sense probe. We adopted dissociated cells, but not sections, for this analysis to avoid overlapping of cells. Hybridized signals were found in approximately 30–50% of the cells in certain developmental stage, and the proportion depended on the preparation of spermatogenic cells and on the type of OR. Figure 5A shows that a single fluorescent dot derived from MOR244-3 mRNAs was observed in nuclei of 50% of the early round spermatids (a total of 173 cells examined). The intensities of the fluorescence were different for various ORs, due to differences in the amount of mRNAs because we observed the same intensities of the OR transcripts in OSNs (Supplementary Fig. S2).



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  		Figure 5  Fluorescent in situ hybridization of multiple probes in isolated round spermatids. Spermatogenic cells isolated from testis were hybridized to a MOR244-3 anti-sense probe or a mixture of anti-sense probes for four ORs expressed in the same stage (MOR244-3, MOR171-31, MOR31-2, and MOR139-3; type B in Figure 3B). (A) The proportion of cells that contain MOR244-3 mRNA signals in round spermatids are shown in the left panel, and representative images of MOR244-3 signals are shown in the right panel. (B) Shown on the left are the proportions of cells containing various numbers of dot-like signals in single round spermatids after hybridization with the mixture of four OR probes, and representative images are shown on the right. The number of fluorescent dots in single cells is indicated beside each cell.

 
Does one testicular cell express one or multiple ORs?

One of the important features of OR gene expression in OSNs is the ‘one cell–one receptor’ rule. In other words, single olfactory neurons selectively express only one of the ~1000 different OR genes (Chess et al. 1994; Ressler et al. 1994; Vassar et al. 1994; Ishii et al. 2001; Serizawa et al. 2003; Lewcock & Reed 2004). To examine whether the ‘one cell–one receptor’ expression pattern also applies to ORs expressed in spermatogenic cells, we conducted in situ hybridization analysis using a mixture of OR probes. Spermatogenic cells isolated from small pieces of the seminiferous tubules were hybridized with a mixture of probes for four ORs that are expressed in early round spermatids (type B in Fig. 3B). Hybridized signals were observed in 86% of early round spermatids (a total of 162 cells examined) (Fig. 5B). Thirty-one percent of spermatids contained more than two fluorescent dots per cell, and the number of fluorescent dots per cell varied from one to four (Fig. 5B). Because each fluorescent dot is derived from a single type of OR, single spermatids appear to express multiple OR genes.

To confirm that the multiple fluorescent signals were derived from different ORs, we performed double-in situ hybridization for two ORs expressed in the same seminiferous tubules. Double-in situ hybridization for MOR171-31 and MOR244-3 (both belong to type B) showed that some proportion of round spermatids expresses two ORs (arrowheads in Fig. 6A). We observed four different types of expression pattern in spermatids (n = 89): only MOR244-3 signals (41%); only MOR171-31 signals (15%); both MOR244-3 and MOR171-31 (14%); and no signal (32%) (Fig. 6B). This means that the proportions of round spermatids expressing MOR244-3 and MOR171-31 are 55% and 29%, respectively, consistent with the results of the one-probe in situ hybridization experiments (i.e. MOR244-3; 50% in Fig. 5A). The proportion of cells expressing both MOR244-3 and MOR171-31 (14%) suggests that the chance of co-expression is solely dependent on random probability. Similar results were obtained from double-in situ hybridization for MOR244-3 and MOR31-2 (n = 116): the proportions of the cells expressing only MOR244-3, only MOR31-2, both ORs, or neither were 37%, 11%, 15%, and 37%, respectively (Fig. 6). These results suggest that single spermatogenic cells can express more than one OR. Because ORs are expressed in only a fraction of the spermatogenic cells, the profile of ORs expressed in each spermatid appears to differ between individual cells.



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  		Figure 6  Double-in situ hybridization of ORs expressed in the same developmental stage during spermatogenesis. Hybridization signals for OR anti-sense probes were detected using the TSA-fluorescein system, and nuclei were counter-stained with DAPI (blue). (A) A representative image of double-in situ hybridization of MOR171-31 (green) and MOR244-3 (magenta) anti-sense probes. Round spermatids containing two signals are indicated by white arrowheads. (B) The numbers of round spermatids containing one OR signal, two signals, or no signal in 10-µm testis sections. (C and D) High-magnification views of the cells containing MOR171-31 and MOR244-3 signals. (E) A representative picture of double-in situ hybridization of MOR31-2 (yellow) and MOR244-3 (magenta). Round spermatids containing two signals are indicated by white arrowheads. (F) Numbers of round spermatids containing one OR signal, two signals, or no signal in 10-µm testis sections. (G and H) High-magnification views of the cells containing MOR31-2 and MOR244-3 signals. Bars, 10 µM.

 
The different expression patterns in the OE and the testis raise a possibility that distinct transcriptional starting sites are utilized in the OE and the testis. Indeed, it has been demonstrated that the transcriptional starting site and exon/intron structure of MOR23 gene are different between in the OE and in the testis (Asai et al. 1996). The transcriptional starting sites of MOR244-3 and MOR171-31 were determined by 5'-RACE analysis, and we found that 5'-UTR structures of these ORs were also different between in the OE and in the testis (Fig. 7). The transcripts isolated from the OE contained one or two exons in 5'-UTR, while those from testis were transcribed from more downstream region and contained no intron.



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  		Figure 7  5'-UTR structures of OR mRNAs in the OE and testis. Exon-intron structures of OR transcripts isolated from the OE and testis are shown. Dark gray boxes indicate the coding region of ORs, and gray boxes indicate 5'-non-coding sequence. The transcripts isolated from the OE contain one or two exons in their upstream-non-coding regions, while those from testis are transcribed from downstream region of the transcription-starting site in the OE and contain no intron. The length of each exon or intron is given in base pairs above each exon or below each line, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Conclusions
 Experimental procedures
 References
 
The present study described developmental expression profiles of ORs expressed in mouse testis. In situ hybridization analysis showed that testicular OR genes were stage-specifically expressed during spermatogenesis, and that the stages of expression were different between ORs. The OR transcripts were localized in a dot-like structure within the nuclei of the spermatogenic cells, suggesting that OR mRNA were accumulated and stored during spermatogenesis. Further, double fluorescent in situ hybridization analysis revealed that single spermatogenic cells can express more than one OR, and that spermatogenic cells are heterogeneous in terms of OR gene expression.

A potential meaning of the stage-specific expression of testicular ORs

We found that the testicular ORs are expressed in three patterns. MOR23, which plays a role in chemosensing and in the regulation of sperm flagellar motility, is transcribed in late round spermatids, just before the cessation of transcription during spermatogenesis (Fukuda et al. 2004). Although the fate of the transcripts for some other OR genes expressed in earlier stages is unclear, the distinct stage-specific expression suggests that ORs may play a role not only in sperm physiology but also in sperm development. For example, MOR248-11, which is expressed in premeiotic cells, is likely to participate in spermatogenesis rather than in sperm physiology. Spermatogenesis is a complex differentiation process that requires the coordinated expression of diverse stage-specific genes. Although the mechanisms underlying the regulation of the coordinate differentiation process have not been elucidated, some peptides and other components secreted by Sertoli cells in combination with follicle stimulating hormone and testosterone are required for germ cell differentiation (Griswold 1995; Sassone-Corsi 1997). Testicular ORs might recognize some hormonal factors at each developmental step and thereby function as regulators of spermatogenesis. However, it remains to be determined whether ORs other than MOR23 are expressed at the protein level and function as chemoreceptors during sperm development.

Post-transcriptional regulation of OR genes in spermatogenic cells

It is particularly interesting that OR gene transcripts accumulated within the nuclei of spermatogenic cells. The dot-like signal is reminiscent of the chromatoid body, which is located near the nucleus in the cytoplasm of spermatogenic cells (Saunders et al. 1992; Oko et al. 1996; Toyooka et al. 2000). Some of the haploid cell-specific gene transcripts and RNA-binding proteins were shown to be localized in the chromatoid body, and it has been postulated that the chromatoid body is involved in the stabilization and translation of germ cell-specific gene transcripts (Noce et al. 2001). Similarly, compartmentalization of OR transcripts in the nuclei of the spermatogenic cells may result in the storage of testicular OR mRNA until the appropriate stage for translation during spermatogenesis.

Significant signals of OR transcripts were not detected in the cytoplasm of the spermatogenic cells in our experiments. Nonetheless, considering that MOR23, which was shown to be functionally expressed in mouse sperm, was also expressed in round spermatids in a dot-like pattern, the transcripts are likely to be transported and translated in the cytoplasm. To detect diffused signals in the cytoplasm, it will be necessary to develop a more sensitive system to detect the low amount of mRNA in situ at the subcellular level, or to construct a specific OR antibody to examine how the transcripts are transported to the cytoplasm and translated into proteins.

A plausible mechanism for one cell-multiple OR expression in spermatogenic cells

In situ hybridization showed that single spermatogenic cells expressed multiple ORs, at least at the mRNA level. Thus, the negative feedback observed in OSNs does not occur in spermatogenic cells. It has been postulated that a negative regulatory signaling is elicited in OSNs by expressed OR proteins, leading to the suppression of all other OR genes (Serizawa et al. 2003; Lewcock & Reed 2004). Because we have not been able to examine the expression of testicular OR protein, it is possible that multiple OR genes are transcribed because there is no translation. However, this is unlikely because multiple OR transcripts were also observed in MOR23 transgenic mice, which have been shown to functionally express a large amount of MOR23 protein in most spermatogenic cells (Fukuda et al. 2004) (data not shown). Different transcriptional initiation sites observed in 5'-race analysis may result in differences in transcriptional regulation, causing distinct patterns of expression in the OE and testis.


   Conclusions
Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
Experimental procedures
 References
 
The following novel findings were demonstrated in this study. First, by examining the developmental expression profiles of OR genes in spermatogenic cells, we found that testicular OR genes are stage-specifically expressed during spermatogenesis and the stages of expression were different between ORs. Second, we found that OR transcripts were localized within the nuclei of the spermatogenic cells and that the signal was concentrated in a dot-like structure. Finally, we explored whether the ‘one cell–one OR’ rule in the olfactory system also applies to spermatogenic cells, and found that, in contrast to the olfactory system, single spermatogenic cells can express multiple ORs at least at the mRNA level. The results suggest that the transcriptional regulation of OR genes in testis is distinct from that in the olfactory system. To determine regulatory role for one cell-multiple OR gene expression as well as the physiological functions of testicular ORs in spermatogenic cells, it will be necessary to examine the protein-level expression of testicular ORs and, further, to identify the endogenous ligands recognized by these ORs.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Experimental procedures
References
 
Cloning of OR genes expressed in testis

Total RNA was prepared from tissues of adult C57BL/6CrSlc mice (Japan SLC) using TRIzol reagent (Life Technologies, Inc.). DNase I (Amplification grade, Invitrogen)-treated RNA was reverse-transcribed using Superscript II (Invitrogen) and NotI-(dT)18 primer (Pharmacia Biotechnology). An equal volume of the DNase I-treated RNA was subjected to a reverse transcription (RT) reaction without Superscript II to confirm that the samples were not contaminated with genomic DNA. The polymerase chain reaction (PCR) was performed in two steps. First the PCR was conducted on the RT mixture using a 5'-primer specific for the target gene and the 3'-NotI primer. A nested PCR was performed on an aliquot (1/50 of the total sample) of the first PCR products with specific primer sets for the target gene. Amplifications were carried out for 35 cycles (94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min) using Ex Taq (Takara) in a total volume of 25 µL. In the amplification using degenerate primers, the annealing temperature of the PCR cycles was set at 45 °C. For screening of testicular OR genes, the specific primers of the following 21 OR genes were constructed from the 5'- and 3'-ends of the coding sequences and used for RT-PCR: MOR281-1, MOR196-2, MOR174-9 (mOR-EG), MOR174-4, MOR9-2, MOR171-17, MOR224-1, MOR139-3, MOR127-2, MOR174-10, MOR174-2, MOR13-6, MOR174-16, MOR174-6/-12, MOR274-1, MOR261-12, MOR248-11, MOR262-7, MOR182-6, MOR114-4, and MOR264-10. Further, specific primers for selected ORs from the microarray results by Zhang et al. (2004b) were constructed and subjected to RT-PCR analysis. Degenerate primers were designed from the amino acids motifs of ORs: MAYDRYVAIC at the end of transmembrane region (TM) 3, KAFSTCSSH at the beginning of TM6, and PMLAPFIY in TM7. These conserved sequence motifs in all known mouse OR genes were aligned, and degenerate primers were designed so that a maximum of OR genes (672) could be theoretically amplified. The degenerate primers are as follows: the upstream primer sequences: 5' ATG KSI TWI GAY MGI TWY GYI GC-3' or 5' TWI GAY MGI TWY GYI GCI RTI TG 3', and the downstream primers: 5' RCA IGT RBI VA (A/G) IGC (C/T) TT 3' or 5' TAI AYI AII GGR TTI A (G/A) C A (T/A) I GG 3'. [R: A and G, W: A and T, S: C and G, B: C, G and T, Y: C and T, M: A and C, K: G and T, V: A, G, and C, I: inosine]. Note that MOR171-31 is encoded by two tandem genes (Olfr1037 and the pseudogene Olfr1036p) on chromosome 2, and in the current studies, we cloned the intact MOR171-31 gene (Olfr1037) from testis cDNA.

In situ hybridization

Digoxigenin (DIG)- or fluorescein isothiocyanate (FITC)-labeled probes were synthesized by in vitro transcription using DIG or FITC RNA labeling mixes (Roche). The full coding sequences of ORs, protamine 1, and H1t in pBlueScript II or pGEM T-vector were used as templates. The primer sequences used to clone cDNA from mouse testis are shown in Supplementary Table S1. The probes used in the hybridization in testis were specific to a particular OR (the sequences did not show more than 80% similarity to other genes) except for MOR139-3, which potentially cross-hybridizes with MOR139-4. In situ hybridization on testis cryosections was performed as previously described (Fukuda et al. 2004). Briefly, cryostat sections (10 µm) of testis from 10-week-old C57BL/6CrSlc mice were postfixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), treated with H2O2/PBS, and acetylated. Sections were then incubated overnight at 65 °C with 200 µL of hybridization buffer (50% formamide, 10 mM Tris-HCl, pH 7.0, 0.2 ng/mL tRNA, 10% dextran sulfate, 1X Denhardt's solution, 600 mM NaCl, 0.25% SDS, 5 mM EDTA) containing ~50 ng of DIG-labeled probe. For double-in situ hybridization, reactions also contained ~50 ng of FITC-labeled probe. The sections were washed twice with 50% formamide in 2x SSC at 65 °C, once with 2x SSC at 65 °C and twice with 0.2x SSC at 65 °C.

For in situ hybridization of isolated spermatogenic cells, small pieces (~3 mm) of seminiferous tubules were treated successively with 0.05% trypsin (Sigma), 0.025% trypsin inhibitor (Sigma), and 0.12 U/µL DNase I (Sigma). The trypsin-treated tubules were washed with PBS after which spermatogenic cells were isolated on to a poly D-lysine-coated glass slide by rolling the pieces of the tubules using elastic glass pipette. The slides were immediately fixed with 4% paraformaldehyde/PBS, treated with H2O2/PBS, acetylated, and subjected to the hybridization procedure.

Detection of hybridization signals

Detection using the TSA biotin system (PerkinElmer Life Science) was performed according to the manufacturer's protocol. Briefly, sections were blocked with 0.5% blocking reagent (TNB) and incubated with horseradish peroxidase (HRP)-conjugated sheep anti-DIG Fab fragment (Roche). Signals were amplified by incubating the sections with biotinyl-tyramide followed by HRP-conjugated streptavidin. The sections were then incubated with 3,3'- diaminobenzidine (Sigma-Aldrich), and counter-stained with methyl green (DAKO).

Detection of double-fluorescent hybridization signals was performed using the TSA plus fluorescence system (PerkinElmer). The slides were blocked with TNB, incubated with HRP-conjugated sheep anti-DIG Fab fragment, and reacted with tyramid-Cy5 solution. After quenching the HRP by incubation for 30 min with 3% H2O2/PBS, the slides were incubated with HRP-conjugated sheep anti-FITC Fab fragment and then reacted with tyramid-Cy3 solution. Finally, sections were counter-stained with DAPI (Molecular Probes).

Hybridized signals in testis cryosections or isolated spermatogenic cells were photographed with TCR SP2 (Leica) or DP70 (Olympus) microscopes. Parameters for fluorescent gain intensities were fixed for all sections. Only fluorescent spots larger than 0.5 µm in diameter were counted as OR mRNA signals.

5' RACE-analysis

Using SMART RACE cDNA Amplification Kit User (Contech Laboratories, Inc.), 5'-RACE was performed according to the manufacturer's protocol. Total RNA purified by Trizol was used as a template. All reaction procedures were performed in a half volume of the manufacturer's protocol.


   Acknowledgements
 
We thank M. Omura for technical advises, R. Niwa for valuable discussion, M. Higurashi for technical help, and all members of the Touhara lab for help and support. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan (PROBRAIN).


   Footnotes
 
Communicated by: Masayuki Yamamoto

* Correspondence: E-mail: touhara{at}k.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Experimental procedures
 References
 
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Received: 2 August 2005
Accepted: 2 October 2005




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