**2. Long-lived transcripts and interacting proteins: role in the sperm maturation during spermiogenesis**

Most transcripts formed in primary spermatocytes are not translated immediately after exiting from the nucleus. These RNAs are reserved in the ribonucleoprotein protein (RNP) complexes for a long time up to the elongation stage during spermatid individualization [6]. In *D. melanogaster*, 529 of the 553 mRNAs detected in spermatid were transcribed in primary spermatocytes [12].

The existence of the NXF (Nuclear eXport Factor) specialized transport receptors, which are the products of paralogous genes of the NXF family and are expressed predominantly in the testes or the brain of humans and mice, has been associated with the presence of long-lived, temporary non-translatable transcripts [13–17]. The most evolutionary conservative member of this family (NXF1) provides transport of the majority of the mRNAs from the nucleus to the cytoplasm [18–21]. The paralogous members of the NXF family differ from the NXF1 protein by divergent sequences enabling interaction with nucleoporins—proteins of nuclear pore complexes and the domain of LRR (leucine-rich repeats). These sequences are responsible for interaction with the partner proteins forming RNP complexes at different steps of mRNA biogenesis [13–15]. The primary localization of testis-specific NXFs in the cytoplasm suggests their participation in the biogenesis of long-lived mRNAs, which are temporarily untranslated [16, 17].

In *D. melanogaster*, the testis-specific *Nxf* paralogous genes are unknown*.* Unlike its orthologous in humans and mice, the *Dm nxf1* (*sbr*) gene does have testisspecific products [22, 23]. The short testis-specific *sbr* transcripts use the alternative promoters in intron 3 and do not include exons 1–3. As a result, the testis-specific truncated SBR protein excludes nuclear localization signals (NLS) present in the canonical SBR protein and in the part of the RNA binding domain (RBD)

#### **Figure 1.**

*The testis-specific* sbr *mRNAs are detected by 5′ and 3′ RACE-PCR.Boxes indicate exons included in the sbr mRNAs. Exon numbers are signed under each box. RBD – RNA-binding domain; LRR – leucine-rich domain; NTF2L – nuclear transport factor 2 like; UBA – ubiquitin associated domain; NLS – nuclear localization signal. In testes, all* sbr *mRNAs have the shortened 3′ UTR.In all transcripts, length of the 3′ UTR varies from 44 to 124 nucleotides. Two* sbr *mRNAs, using the alternative promoters in intron 3, give rise the shortened protein isoform without NLS and the part of RBD, which are present in the canonical full-length SBR (published in Mamon et al. [24]).*

**153**

**Figure 2.**

*Spermatogenesis in* Drosophila melanogaster: *Key Features and the Role of the NXF1…*

(**Figure 1**) [24]. Such truncated SBR form can have other functions in addition to

*Adult testis immunofluorescence using anti-SBR (Alexa 546 goat anti-mouse). In the prophase of meiosis (arrowhead) and at the round spermatid stage (arrow)—The SBR proteins are primarily localized in the* 

During transcriptional activities—the prophase of meiosis and the round spermatid phase—the SBR protein is primarily found in the nucleus (**Figures 2, 3**). This is coordinated with the known universal function of NXF1 proteins in various organisms [18–21]. Unfortunately, antibodies to C-terminal part of the SBR protein

Nuclei of spermatids in onion stage are brightly painted fluorochromes conjugated with anti-SBR (**Figure 2**). Nebenkern is near each of nucleus and is free from SBR. Only 24 genes are transcribed in *Drosophila* spermatids [12], but spermatid nuclei show the brightest coloring by anti-SBR. Why at onion stage such high concentration of transport receptors for mRNA is necessary. It is possible that spermatid nuclei may be one of the docking place for some long-lived mRNAs because the most dramatic events depending from translation of these mRNAs are related to nucleus and/or structures connected with nucleus during the sperm maturation. In elongated spermatids, the SBR protein is localized on a nuclear surface asymmetrically, only along one of its sides (**Figure 4**). Spermatid elongation is period of intensive nuclear morphogenesis [1]. The nuclear envelope becomes asymmetrical, with nuclear pores being disposed along one side of elongated nucleus. In this region, the dense body consisting from a microtubule (MT) and actin-rich structure is formed and also localized along one side of the nucleus [25]. Nucleoporin binding domains (NTF2L and UBA) in the SBR proteins (**Figure 1**) allow them to participate in the storing of the RNP complexes with the long-lived RNAs connected with the nucleoporins on the nuclear envelope. After the RNP complexes dissociate, the translation of their mRNAs begins close to the nucleus, where there is a need for appropriate proteins. Then the components of the RNP complexes, including the SBR proteins,

the nuclear RNA export, which are important for spermatogenesis.

*nucleus. Asterisk denotes the stem cells zone. Scale bar, 75 μm.*

cannot contradistinguish full-sized and truncated SBR isoforms.

*DOI: http://dx.doi.org/10.5772/intechopen.90917*

*Spermatogenesis in* Drosophila melanogaster: *Key Features and the Role of the NXF1… DOI: http://dx.doi.org/10.5772/intechopen.90917*

#### **Figure 2.**

*Animal Models in Medicine and Biology*

important component of these RNP-complexes.

**maturation during spermiogenesis**

in spermatid were transcribed in primary spermatocytes [12].

mRNAs, which are temporarily untranslated [16, 17].

actin structure, termed the investment cone, forms a new membrane [10, 11]. Each cell becomes independent and turns into a mobile spermatozoon. Development of a mature spermatozoon with the change of histones on protamines in nucleus, the building of an acrosome and a tail, the forming of cellular membrane, occurs without transcription owing to translation of the long-lived mRNAs. Such mRNAs are keeping as a part of the RNP-complexes localized in the different cellular compartments. The testis-specific isoform of the Dm NXF1 (SBR) protein can be an

**2. Long-lived transcripts and interacting proteins: role in the sperm** 

The existence of the NXF (Nuclear eXport Factor) specialized transport receptors, which are the products of paralogous genes of the NXF family and are expressed predominantly in the testes or the brain of humans and mice, has been associated with the presence of long-lived, temporary non-translatable transcripts [13–17]. The most evolutionary conservative member of this family (NXF1)

provides transport of the majority of the mRNAs from the nucleus to the cytoplasm [18–21]. The paralogous members of the NXF family differ from the NXF1 protein by divergent sequences enabling interaction with nucleoporins—proteins of nuclear pore complexes and the domain of LRR (leucine-rich repeats). These sequences are responsible for interaction with the partner proteins forming RNP complexes at different steps of mRNA biogenesis [13–15]. The primary localization of testis-specific NXFs in the cytoplasm suggests their participation in the biogenesis of long-lived

In *D. melanogaster*, the testis-specific *Nxf* paralogous genes are unknown*.* Unlike

specific products [22, 23]. The short testis-specific *sbr* transcripts use the alternative promoters in intron 3 and do not include exons 1–3. As a result, the testis-specific truncated SBR protein excludes nuclear localization signals (NLS) present in the canonical SBR protein and in the part of the RNA binding domain (RBD)

its orthologous in humans and mice, the *Dm nxf1* (*sbr*) gene does have testis-

Most transcripts formed in primary spermatocytes are not translated immediately after exiting from the nucleus. These RNAs are reserved in the ribonucleoprotein protein (RNP) complexes for a long time up to the elongation stage during spermatid individualization [6]. In *D. melanogaster*, 529 of the 553 mRNAs detected

**152**

**Figure 1.**

*(published in Mamon et al. [24]).*

*The testis-specific* sbr *mRNAs are detected by 5′ and 3′ RACE-PCR.Boxes indicate exons included in the sbr mRNAs. Exon numbers are signed under each box. RBD – RNA-binding domain; LRR – leucine-rich domain; NTF2L – nuclear transport factor 2 like; UBA – ubiquitin associated domain; NLS – nuclear localization signal. In testes, all* sbr *mRNAs have the shortened 3′ UTR.In all transcripts, length of the 3′ UTR varies from 44 to 124 nucleotides. Two* sbr *mRNAs, using the alternative promoters in intron 3, give rise the shortened protein isoform without NLS and the part of RBD, which are present in the canonical full-length SBR* 

*Adult testis immunofluorescence using anti-SBR (Alexa 546 goat anti-mouse). In the prophase of meiosis (arrowhead) and at the round spermatid stage (arrow)—The SBR proteins are primarily localized in the nucleus. Asterisk denotes the stem cells zone. Scale bar, 75 μm.*

(**Figure 1**) [24]. Such truncated SBR form can have other functions in addition to the nuclear RNA export, which are important for spermatogenesis.

During transcriptional activities—the prophase of meiosis and the round spermatid phase—the SBR protein is primarily found in the nucleus (**Figures 2, 3**). This is coordinated with the known universal function of NXF1 proteins in various organisms [18–21]. Unfortunately, antibodies to C-terminal part of the SBR protein cannot contradistinguish full-sized and truncated SBR isoforms.

Nuclei of spermatids in onion stage are brightly painted fluorochromes conjugated with anti-SBR (**Figure 2**). Nebenkern is near each of nucleus and is free from SBR. Only 24 genes are transcribed in *Drosophila* spermatids [12], but spermatid nuclei show the brightest coloring by anti-SBR. Why at onion stage such high concentration of transport receptors for mRNA is necessary. It is possible that spermatid nuclei may be one of the docking place for some long-lived mRNAs because the most dramatic events depending from translation of these mRNAs are related to nucleus and/or structures connected with nucleus during the sperm maturation. In elongated spermatids, the SBR protein is localized on a nuclear surface asymmetrically, only along one of its sides (**Figure 4**). Spermatid elongation is period of intensive nuclear morphogenesis [1]. The nuclear envelope becomes asymmetrical, with nuclear pores being disposed along one side of elongated nucleus. In this region, the dense body consisting from a microtubule (MT) and actin-rich structure is formed and also localized along one side of the nucleus [25]. Nucleoporin binding domains (NTF2L and UBA) in the SBR proteins (**Figure 1**) allow them to participate in the storing of the RNP complexes with the long-lived RNAs connected with the nucleoporins on the nuclear envelope. After the RNP complexes dissociate, the translation of their mRNAs begins close to the nucleus, where there is a need for appropriate proteins. Then the components of the RNP complexes, including the SBR proteins,

#### **Figure 3.**

*Prepupal testis immunofluorescence using anti-SBR (Alexa 488 goat anti-mouse – A, B) and DAPI (A′, B′). A. Most part of the future gonad is represented of growing spermatocytes, primary spermatocytes, and meiocytes. Initially, the shape of the nucleus is round (A, thin arrow); then the shape becomes irregular (A, short arrow). B. Cyst of 16 meiocytes with the cytoplasmic localization of the SBR protein. Bivalents partially condensed (arrows). Asterisk denotes the stem cells zone. Scale bar, 75 μm.*

#### **Figure 4.**

*Spermatids at the elongation or individualization stages in* D. melanogaster *males (Alexa Fluor 546). Spermatids at the elongation stage: the SBR protein moves onto one side of the spermatid nuclear. The SBR protein leaves the condensed nuclei and is translocated in large granules into the formed spermatozoon tails at the final stage of spermatid elongation before individualization. 63× magnification.*

**155**

*Spermatogenesis in* Drosophila melanogaster: *Key Features and the Role of the NXF1…*

elimination of SBR during spermatid individualization (**Figure 4**).

relocate in a spermatozoon tail as the granules and are degradate (**Figure 4**) [26, 27]. Proteasome degradation of the proteins is necessary for the formation of mature sperm [28]. SBR (Dm NXF1) protein has UBA-like domain (**Figure 1**), which are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells [29, 30]. Proteasome degradation is important for cell cycle progression [31]. Testisspecific proteasome degradation in *D. melanogaster* probably provides effective

It is important that mRNAs encoded by all of the post-meiotically transcribed

A feature of spermatogenesis is that many genes produce shortened transcripts that encode shortened proteins. These proteins cannot function as the full-sized proteins encoded by the same genes [32]. This is due to the presence of testisspecific promoters or alternative splicing variants [33]. In addition, testis-specific transcripts often use an alternative polyadenylation site located upstream, compared to those in the transcripts of the same gene in other organs [33, 34]. This may be important for the regulation of translation and degradation of transcripts during spermatogenesis. All the transcripts of the *Dm nxf1* (*sbr*) gene have the shortened

genes are localized to the extreme distal ends of elongated spermatids. These mRNAs are subdivided into two classes named by comet and cup transcripts [12]. There is not a concentration of SBR to the distal ends of elongated spermatids.

**3. Allele-specific effects of the** *sbr* **gene including the male infertility**

*Dp (1;Y)y + v +* males. *Dp (1;Y)y + v +* is the duplication of the segment of the X chromosome with the *sbr+*, y + and v + alleles, translocated on the Y chromosome. The duplication compensates a lethal effect of the different alleles of the *sbr* gene localized on X-chromosome [39]. *sbr10* is the thermosensitive allele with a block of the heat shock protein synthesis under heat shock (HS) [40]. It is a recessive feature of the *sbr10* allele [38]. Adult males carrying the *sbr10* allele are thermosensitive and die from 37°C, 1 h [8]. HS increased the number of abnormalities, of not only paternal but also maternal sex chromosome sets, in the offspring of the *sbr10* males. Meiocytes

It was interesting to know what feature is characterized the SBR protein distribution in the future gonads of *D. melanogaster* males on the pupal stage. On a pre-pupa stage of development, the future gonad in *D. melanogaster* males is spherical with two poles with dividing cells (**Figure 3**). One pole produces germ-line cells and

Mutations of the house-keeping genes are characterized by a broad range of pleiotropic effects. It is no surprise that mutations of the *Dm nxf1* gene lead to both male and female sterility and increase the frequency of the development malformations (FlyBase [35]). That may be the result of disruption of the NXF1 general functions. Allele-specific effects of mutations in the *Dm nxf1* gene suggest that its products may have specialized functions. The organ-specific products of *sbr* can provide such specialization [22–24]. In addition, the source of specialized functions may be the presence of functionally significant sequences in the SBR protein, responsible for interaction with the certain partners. As a result, SBR may be involved in other processes than the nuclear-cytoplasmic export of mRNAs [36, 37]. A mutant product that disrupts the system of macromolecular interactions can have a dominant negative effect, in this case heterozygosity at the null allele will be better than the presence of a mutant protein along with the normal product [38]. Among the mutant alleles of the *sbr (Dm nxf1)* gene with the dominant effects are the formation of three-pole

*/+* females [36] and sterility of *sbr<sup>12</sup>/*

*DOI: http://dx.doi.org/10.5772/intechopen.90917*

3′UTR in testes [22, 23].

spindles during the first meiotic division in *sbr<sup>5</sup>*

were the thermosensitive stage for this unexpected effect [8].

*Spermatogenesis in* Drosophila melanogaster: *Key Features and the Role of the NXF1… DOI: http://dx.doi.org/10.5772/intechopen.90917*

relocate in a spermatozoon tail as the granules and are degradate (**Figure 4**) [26, 27]. Proteasome degradation of the proteins is necessary for the formation of mature sperm [28]. SBR (Dm NXF1) protein has UBA-like domain (**Figure 1**), which are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells [29, 30]. Proteasome degradation is important for cell cycle progression [31]. Testisspecific proteasome degradation in *D. melanogaster* probably provides effective elimination of SBR during spermatid individualization (**Figure 4**).

It is important that mRNAs encoded by all of the post-meiotically transcribed genes are localized to the extreme distal ends of elongated spermatids. These mRNAs are subdivided into two classes named by comet and cup transcripts [12]. There is not a concentration of SBR to the distal ends of elongated spermatids.

A feature of spermatogenesis is that many genes produce shortened transcripts that encode shortened proteins. These proteins cannot function as the full-sized proteins encoded by the same genes [32]. This is due to the presence of testisspecific promoters or alternative splicing variants [33]. In addition, testis-specific transcripts often use an alternative polyadenylation site located upstream, compared to those in the transcripts of the same gene in other organs [33, 34]. This may be important for the regulation of translation and degradation of transcripts during spermatogenesis. All the transcripts of the *Dm nxf1* (*sbr*) gene have the shortened 3′UTR in testes [22, 23].
