Now there is interest in finding factors that, once in the ovum, can affect the development of offspring. The sbr (Dm nxf1) gene in D. melanogaster belongs to the evolutionarily conserved family of nxf (nuclear export factor). It is involved in controlling of male fertility and forming the factor that affects the segregation of maternal and paternal chromosomes after fertilization. The Dm NXF1 (SBR) protein seems to play a role in forming this mysterious factor during the meiotic period of spermatogenesis. Male germ cells develop as a syncytium, and success of spermatid individualization depends, to a great degree, on gene expression in primary spermatocytes. Most transcripts formed in primary spermatocytes are long-lived. The presence of the Dm NXF1 protein in the nucleus as well as in the cytoplasm suggests that it plays a role in the biogenesis of long-lived RNA and in cytoskeletal reorganization.
- RNA-binding protein
Spermatogenesis is an evolutionary conservative process. In testes, the developing germline cells divide synchronously and with incomplete cytokinesis remaining interconnected by cytoplasmic bridges. Therefore, the germline cells develop as a syncytium and only become separated from each other at the end of spermatogenesis during spermatid individualization. In
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 . In
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, 14, 15, 16, 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, 19, 20, 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, 14, 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].
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, 19, 20, 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
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 . 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 . This is due to the presence of testis-specific promoters or alternative splicing variants . 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
3. Allele-specific effects of the
sbrgene including the male infertility
Mutations of the house-keeping genes are characterized by a broad range of pleiotropic effects. It is no surprise that mutations of the
It was interesting to know what feature is characterized the SBR protein distribution in the future gonads of
As the growing spermatocytes are characterized by the high level of transcriptional activity, the long-lived RNAs seem to be the most appropriate candidate for the role of factors that, along with the spermatozoon, enter an ovum during fertilization and can participate in chromosome disjunction. Due to the process of translation, a small number of these molecules are sufficient for the regulation of chromosome segregation. This is particularly important during the first hours of embryonal development of
One more target of
4. The centrosome for male meiosis and building of the axoneme
The centrosome plays a special role in spermatogenesis. It determines the polarity of the stem cells, maintaining contact with the hub and enabling the asymmetric division of stem cells . Male spermatocytes of the wild type contain two centrosomes each. Each centrosome has two orthogonal centrioles that are 10 times larger than those in other cells . Thus, during meiosis I, each of two centrosomes contains a pair of duplicated centrioles.
No centriole duplication occurs before meiosis II. Before chromatid segregation in secondary spermatocytes, each pair of centrioles divides into two single centrioles. Prior to chromatid segregation during meiosis II, one of the centrioles migrates to the opposite pole of a cell. As a result, each spermatozoon inherits only one centriole, which becomes the basal body forming the axoneme [1, 52]. Paternal centrosome participates in the formation of astral microtubules for moving male and female pronuclei toward each other . The role of centrosome RNAs in the asymmetry distribution of cytoplasmic determinants among daughter cells  makes plausible the hypothesis that centrosome may be a carrier of the paternal factors, affecting the embryo development.
The axoneme growth during sperm maturation needs translation of the long-lived mRNAs coding the components of axonemal complex. Morphological defects of axoneme are a characteristic dominant manifestation of
Translational control is crucial for morphogenical events that take place in the absence of transcription during spermiogenesis [26, 54]. The transcripts encoding proteins required for post-meiotic processes including spermiogenesis are almost all abundantly transcribed in spermatocytes . In
5. Incomplete cytokinesis providing a cellular communication as a feature of male generative cells
Syncytial development during the formation of male generative cells is conservative and is characteristic for different animals [59, 60]. It is assumed that the link between the cells in the syncytium is important primarily for synchronizing differentiation processes . The cytoskeleton and the molecules providing a cellular communication are the main components supporting of equality of each cell in the syncytium.
The cytokinesis of spermatogenic cells is characterized by the formation of ring canals linking the cells that have undergone meiosis or mitosis . During cell division, a contractile ring, made of actin and myosin II filaments, assembles as a result of the interaction between the plus ends of the microtubules and the cell cortex . The contractile ring is double-sided. The microtubules whose minus ends directed toward centrosome on one pole of the dividing cell bind to one side of the contractile ring by their plus ends. The microtubules that have their minus ends turned toward centrosome on the other pole of the dividing cell bind to the opposite side of the contractile ring. Thus, the ring attached equatorially to the membrane of the dividing cell forms an actin-myosin cleavage furrow . The ingression of the cleavage furrow is accompanied by the growth of the daughter cell membranes.
+TIPs—complexes linked to the plus-ends of the microtubules—play a special role in the interaction of the plus-ends of the microtubules with the cell cortex . +TIPs also play an important role in finding and capturing microtubule targets—the cortex and the chromosomes [64, 65]. Localized mRNAs are anchored at the plus ends of microtubules especially in polarized cells . SBR also may be one of the factors that interact with the plus-ends of microtubules.
It is worth noting that the Hs NXF1 (TAP) factor in humans was initially identified
Tyrosine phosphorylation is important for regulating the assembly and disassembly of the actin cytoskeleton at cell-cell junctions. In many cases, tyrosine phosphorylation in proteins of the junction complex—plakoglobin and β-catenin in adherens junctions—disrupts interactions between cytoskeleton and membrane . It is believed  that tyrosine phosphorylation in targets leads to the disassociation of phosphorylated actin from the contractile ring that forms during the cytokinesis of the cells comprising the syncytium during spermatogenesis. This stabilizes the interaction between the walls of the ring and the cell membrane, ends cell division, and forms the ring canal . Building a new cell wall at the cleavage site during cytokinesis requires the involvement of components of the cell membrane and the signaling molecules . A system of transport molecules ensures the delivery of the necessary elements to the region where the cell membrane is being formed .
Interactions of NXF1 with cytoskeleton  and the cellular membrane  may help finding partners of NXF1 in the cytoplasm. Not surprisingly, the closest evolutionary relative of NXF1 is dynein. Dynein plays a significant role in cytokinesis, chromosome segregation, and in enabling the movement of the flagellum of the sperm [70, 71].
6. Homology of proteins of the NXF family and axonemal dyneins as a source of evolutionary relationship
Searching for related proteins and aligning sequences using the multiple sequence alignment tool base has demonstrated that among the proteins that are homologous to the factor NXF1, axonemal dynein of various organisms—especially its light chain—is most frequent. Aligning sequences that exhibit homology demonstrates that the N-terminus of the Dm NXF1 protein, which includes RBD domains and the LRR (leucine-rich repeats), corresponds to the axonemal light chain of dynein in vertebrates (Figure 6). At the LRR site, there is a sequence that exhibits a high degree of homology (marked by bold typeface in Figure 6), both when comparing different NXF factors (orthologous and paralogous), and comparing the SBR protein with sequences of light chains of dynein (Figure 6). The functions of this sequence are yet to be determined.
The dynein complex is a multicomponent system consisting of light, light intermediate, intermediate, and heavy chains of dynein . Interacting with a multitude of protein and ribonucleic partners, the LC8 and TcTex1 light chains of dynein enable a link between different forms of the transported cargo and heavy chains of dynein that function as molecular motors [73, 74]. By interacting with its partner dynacin complex, the dynein complex enables cytoplasmic transport of macromolecular complexes, vesicles, and organelles , including particles containing RNAs [74, 76].
In the fruit fly, the null mutants of the genes that encode the light or heavy chain of dynein have anomalies of wings and bristles, exhibit male and female sterility, and are characterized by disrupted sensory axon trajectories , thus demonstrating a similarity to the mutants of the
During spermatid individualization, the dynein light chains 1 (DLC1) participate in the assembly of the F-actin . Dynein is necessary for centrosome separation, for forming the division spindle, and for aligning chromosomes equatorially during metaphase [78, 79]. During the interaction with the surface of microtubules, dynein also interacts with MAP proteins . During prometaphase, the DYNLT3 light chain can be observed in kinetochores. During metaphase, dynein moves toward the spindle pole [81, 82]. The dynein complex leaves the kinetochores along with checkpoint proteins. This could be related to the transport of checkpoint proteins (for example, Bub3) from kinetochores to spindle poles . If we recall the homology of the proteins Bub3 and Rae1  and the fact that the protein Rae1, found in RNP complexes that are important for forming the spindle, is capable of direct interaction with NXF1 , it is possible to consider these factors as polyfunctional. They are taking part both in nuclear-cytoplasmic transport of macromolecules and in the formation of the mitotic apparatus of the cell.
Because it is the light chain of dynein in the dynein complex that is responsible for interaction with the cargo , this resemblance to the N-terminus of the SBR protein suggests that SBR may have the same partners as a dynein complex (that these proteins can have the general partners or the general functions, and they are evolutionary conservative).
We are grateful to the staff of KHROMAS (the Center of study of ultrastructure and molecular composition of biological objects at Biological Faculty of St. Petersburg State University) and to its head Elena R. Gaginskaya for their most important assistance in performance of this work.
The Russian Foundation for Basic Research supported the work (project 19-04-01255).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.