The Y chromosome is a sex chromosome found in males of different groups of animals, including mammals and Diptera. Whereas in mammals the development of an organism according to the male type is determined by the presence of a functional Y chromosome, in Drosophila, sex is determined by the ratio of the number of X chromosomes to the number of the autosomes: normally, the presence of two X chromosomes triggers development according to the female type, and one—according to the male type ^[8][9]. Thus, individuals with the XXY genotype are female in flies and male in mammals, while X0 are female in mammals and male in flies. In Drosophila with the X0 karyotype, there are no severe structural or functional body disorders, except for male sterility ^[6]. Fly Y chromosome is not involved in sex determination. Several genes functioning in sex determination of flies, such as Sex-lethal (Sxl), transformer (tra), transformer-2 (tra2), and others, have been found to date. Sex-specific mRNA splicing of major Drosophila sex-determining genes is a complex process providing female-specific transcripts triggering female-type development ^[8][9]. In Diptera, the Y chromosomes arose from the autosomes repeatedly (Figure 2a), which provides good material for studying parallel processes of convergent evolutionary development ^[10][11][12]. While the gene content of the autosomes in Diptera is conserved and originates from a common ancestor, gene composition of the Y chromosomes varies significantly, and the rate of acquisition of new genes often significantly exceeds the rate of their loss ^[13]. In Diptera, the transfer of genes from other chromosomes contributes to the evolution of the Y chromosome. Relatively young, newly formed Y chromosomes of flies, being formed from tens of thousands of years to a few million years ago, maintain the structure and genes of the ancestral autosome, while most of the genes on the old fly Y chromosomes have been acquired subsequently due to a transfer from the autosomes or the X chromosome. Old Y chromosomes such as in D. melanogaster that presumably have been persisted for long time (several decades of millions of years) are often highly heterochromatic, contain a large amount of repetitive DNA, and their genes undergo degeneration ^[10][12].

Drosophila miranda contains a pair of young neo-sex chromosomes that were born ~1.5 million years ago (MYA) after splitting from the closely related species Drosophila pseudoobscura. D. miranda is an excellent model for studying early sex chromosome differentiation given the appearance of new advanced methods of genomic sequencing and assembly. Its neo-sex chromosomes have been created by the fusion of the former autosome 3 with the ancestral, degenerated Y chromosome of this clade (Figure 2b) ^[14]. Initial stages of Y evolution are characterized by massive amplification of distinct classes of genes. The neo-Y chromosome of D. miranda initially contained about 3000 protein-coding genes, but during its evolution, it has acquired over 3200 genes, primarily by tandem amplification of protein-coding genes that were ancestrally present on the chromosome. Testis-specific and dosage-sensitive genes appear to have amplified and have been fixed on the neo-Y to facilitate male fitness. The neo-X and neo-Y chromosomes in D. miranda still maintain a high homology across their length, with up to ~98% sequence identity in homologous regions ^[14][15]. A cohort of meiosis-related multi-copy Y-linked genes have independently co-amplified on the X, and their expansion is probably driven by conflicts over segregation. These co-amplified X-Y genes are highly expressed in spermatogenesis and are significantly enriched for meiosis-related and RNA interference functions. Drive systems on the sex chromosomes presumably include distorter elements causing meiotic drive and biasing progeny sex ratio, and suppressor elements evolving on the opposite chromosome for preventing meiotic drive ^[16]. It has been proposed that their co-amplification is driven by X/Y antagonism providing increased transmission of a certain sex chromosome to the next generation, where suppression of chromosome can be mediated by sequence homology between the suppressor and distorter elements with participation of the RNA interference pathways. It has been suggested that newly emerged sex chromosomes are a battleground for meiotic drive and X-Y inter-chromosomal conflicts ^[14][16].

In some Drosophila species, such as D. pseudoobscura, the old Y chromosome has become part of the autosome, while the neo-Y has been presumably formed de novo several MYA (Figure 2a) ^[12][17]. At the same time, the gene content of the Y chromosomes in D. pseudoobscura and D. melanogaster is completely different, except that both species maintain various Y-linked genes necessary for male fertility. The same pattern can be seen upon comparison of other Diptera species: despite the different evolutionary history, the functions of most Y-chromosomal genes are related to the male reproductive system ^[5][12]. A recent report about D. willistoni confirms the previously observed Y duplication of autosomal or X-linked testis-specific genes and the prominent role of gene gains in the evolution of Drosophila Y chromosomes ^[18].

In many animals, sex is determined by a pair of heteromorphic X and Y chromosomes. According to modern concepts, sex chromosomes originate from an ancestral pair of autosomes, one of which acquires a sex-specific gene, which starts the process of differentiation of the sex chromosomes. In mammals, this event occurred only once in the common ancestor of marsupials and placentals prior to their splitting, about 160–180 MYA ^{[19][20][21][22]}. The proto-Y chromosome of all mammals (from kangaroo to human) arose from a single autosome in which one of the alleles of the SOX3 gene, as a result of a mutation, became the sex-determining gene SRY ^[23][24]. It is important to note that this gene is not responsible for all sex characteristics alone. The product of the sex determination gene only provides a switch, triggering a certain pathway of development. Unique evolutionary forces facilitated the selection and accumulation of male-beneficial mutations around the SRY locus, and the linkage between them was supported by selective pressure to avoid crossing over between the proto-Y and proto-X ^[25]. As a rule, if the dominant allele causes the development of a male, then the chromosome in which it is located becomes the Y chromosome (and its homolog is called the X). In birds, males are the homogametic sex (ZZ) and females are the heterogametic (ZW) ^[21]. Currently two main hypotheses about sex determination in birds are presented. One of them postulates the presence of the key gene controlling ovarian development or inhibiting testis differentiation in the W chromosome, while the other one proposes the number of Z chromosomes as a key sex-determining factor ^[21][26]. In last case, sex determination is thought to be provided by a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosomal gene DMRT1 encoding transcription factor. Recently it was shown that male chicken (ZZ) with a single functional copy of DMRT1 (other was deleted by a CRISPR-Cas9-based monoallelic targeting approach) developed ovaries in place of testes. It indicated that DMRT1 is the key sex determination switch in birds essential for testis development ^[26]. In addition, it was found that the synthesis of estrogen is also an essential factor in primary sex determination in chicken, and that estrogen production is controlled by expression of DMRT1 ^[26]. These data support the second hypothesis that the dosage of genes on the Z chromosome determines the sexual differentiation in birds ^[26].

The loss of recombination leads to the inefficiency of natural selection and causes the ensuing accumulation of Y-linked loss-of-function mutations, chromosome-wide gene decay, and amplification of repetitive DNAs ^{[27][28][29][30]}. In parallel to the loss of genes, Y chromosomes have accumulated large amounts of DNA repeats, and the D. melanogaster old Y chromosome mainly consists of heterochromatin (Figure 2a) ^[4][21]. Despite the human Y chromosome having undergone a rapid decay early in evolution, its massive degeneration then dramatically stopped. Genes that remained intact currently show remarkable stability, and no human Y-linked genes have been lost during the last 44 million years ^[22][31]. The maintenance of human Y-linked genes is mainly associated with two functional categories: genes essential for male reproductive functions and dosage-sensitive ubiquitous housekeepers ^[32]. Studies of males with Y deletions have allowed researchers to identify three ‘azoospermia factor’ (AZF) regions, AZFa, AZFb, and AZFc, and partially map within them the genes essential for spermatogenesis ^[33]. The AZFa deletions affecting the DBY gene cause the most severe azoospermia phenotype, exhibiting a complete loss of testis germline cells accompanied by the maintenance of somatic Sertoli cells (the so-called Sertoli Cell-Only Syndrome; SCOS) ^[34][35][36].

As in fruit flies, mammalian Y chromosomes also exhibit gene amplification, with the amplicon structures predominantly containing genes with testis-specific functions ^[37]. The structure of such genes is maintained by intra-chromosomal gene conversion. The amplicon region of the human Y chromosome contains eight massive palindromes ranging in length from 9 kb to 1.45 Mb with nucleotide identity of the arms over 99.9% ^[38][39][40]. Due to the presence of repeating structures, local intra-chromosomal gene conversion is possible, as well as intra- and inter-chromatid exchange. These mechanisms partially compensate for the lack of recombination with the X chromosome by eliminating harmful mutations. At the same time, inter-chromatid recombination can in some cases lead to the formation of isodicentric chromosomes formed by homologous crossing over between opposing arms of palindromes on sister chromatids. This may be accompanied by the loss of certain regions containing genes essential for spermatogenesis, and in some cases can lead to the loss of the Y chromosome during cell division with clinical consequences ranging from spermatogenic failure to sex reversal and Turner syndrome ^[41].

The loss of the ability to recombine plays a key role in establishing the structure of the Y chromosome, because recombination could lead to a disruption of sex determination and the formation of infertile intermediate variants ^[30][42]. Conversely, mutations that prevent recombination between proto-X and proto-Y, such as inversions, deletions, or accumulation of repeats, are supported by selection. Reducing the ability of recombination with the homologous X chromosome dramatically accelerated the evolution of the Y chromosome preventing the elimination of emerging mutations via crossing over, while the X chromosome has retained the ability to cross over in the homogametic sex. This led to the degeneration of most of the original Y-chromosomal genes, and multiple deletions caused a significant size decrease with a relative increase in the proportion of non-coding heterochromatic regions. The rapid evolutionary degeneration of the Y chromosomes, typical in a wide range of species, leads to the hypothesis that in the future the human Y chromosome may disappear altogether. This hypothesis is based not only on extrapolation, but is also indirectly supported by precedents in the evolution of some species including multiple fishes, reptiles, grasshoppers, cockroaches, and dragonflies ^[43][44][45]. However, other researchers claim that human Y degeneration stopped millions of years ago and currently nothing threatens Y chromosome survival ^[46].

As a rule, a single gene copy appears to be enough to provide development and life-cycle maintenance of diploid animals; however, a small cohort of genes exhibits a high sensitivity in case of decreased gene dosage. This phenomenon is known as haploinsufficiency, and it is associated with many developmental disorders in human ^[47][48][49]. Comparing the evolution of flies and humans, one could assume that the Y-chromosomal genes, which have homologues on the X chromosome and do not directly contribute to the functioning of the male reproductive system, are relics that will disappear over time, as has apparently happened in flies. However, their maintenance can be determined by the peculiarities of the dosage compensation system in mammals. In male flies, the genes of the only X chromosome are overactivated in somatic tissues, eliminating the problem of haploinsufficiency and potentially lethal imbalance between the X and autosome transcriptional level in the two sexes. Therefore, in flies, X activation may eventually compensate for haploinsufficient homologous genes lost on Y, which is impossible in mammals. In contrast, in female mammals, inactivation of one of the two X chromosomes occurs. However, according to various estimates and in distinct types of human cells, 20–30% of genes of inactive X chromosome escape the inactivation ^[50][51]. In mammals, haploinsufficient Y-chromosomal genes have X-chromosomal homologues that avoid inactivation during dosage compensation in females, which indicates the need for their expression on both sex chromosomes to ensure normal functions in the body. Thus, in males, these dosage-sensitive genes cannot disappear from the Y chromosome without negative consequences, and they can survive under selective pressure ^[31]. This hypothesis is supported by the maintenance of functional X-Y gene pairs associated with housekeeping regulatory functions such as lysine demethylation, stem cell self-renewal, splicing, translation initiation, and deubiquitylation ^{[31][32][50][52]}. Strict dosage requirements for sex-linked genes are demonstrated in the case of Turner syndrome (exhibiting X0 karyotype or mosaicism) and Klinefelter syndrome (XXY), since such genes have been haploinsufficient or overexpressed, respectively, in these karyotypes ^[51]. Turner syndrome is a genetic condition caused by complete or partial loss of the second sex chromosome in human. Half the patients with Turner syndrome have the X0 karyotype (monosomy of the X chromosome), the other half exhibits mosaicism or a presence of the fragmented X or Y chromosomal material and other more complex karyotypes ^[53][54]. Studies of manifestations of this syndrome indicate that the functions of the Y chromosome consist not only of ensuring the normal functioning of the male reproductive system. Due to the absence of the SRY gene, which is the key to triggering male-type development, patients with this syndrome are exclusively female, with multiple body disorders: small size, rudimentary ovaries and infertility, pathologies of the cardiovascular system, autoimmune disorders, increased risk of developing diabetes, and cognitive impairment ^[54]. Individuals with Klinefelter syndrome are infertile as a result of excess gene dosage of X escape genes, and abnormal meiotic pairing of the sex chromosomes. An atypical number of X or Y chromosomes (XXY, XXX, or X) contributes to spatial chromosome conformation changes and leads to disruption of DNA methylation patterns of autosomal genes, causing distinct disease phenotypes: mental illness, cancer, and disrupted fertility ^[51].

Despite their independent evolutionary origins in different species, Y chromosomes in species with heterogametic males have a number of similar features: they are usually smaller than X chromosomes, contain significantly fewer genes, most of which are related to the male reproductive system, and also have a relatively large number of repeats and significant areas occupied by heterochromatin. It is worth noting that in species with heterogametic females, such as birds, the sex-specific chromosome W also resembles the Y chromosome in structure, and is characterized by relatively small size, heterochromatinization, and fewer genes ^[21]. Such common patterns indicate the convergently evolved structural features of these chromosomes. It has been proposed that such convergent evolution is due to the similar nature of the selection pressure. Another common feature—the acquisition of repetitive sequences and the loss of most of the original genes—is associated with accelerated Y evolution due to the loss of recombination with the X chromosome ^[27]. The difference between the evolution of the Y chromosome in mammals and Diptera is mainly that in Diptera the acquisition of new genes often significantly prevails over the loss of the original ones; although, both processes take place in both groups. Presumably due to slower changes in mammals, the evolutionary processes have not yet reached the point where the Y chromosome has lost all homology with the X chromosome.

Attempts to understand how these patterns are generated can be important not only for fundamental evolutionary biology, but also for biomedical challenges, since Y-chromosomal pathologies in humans differ from other genetic anomalies due to the unique nature of the Y chromosome. It is convenient to study the Y chromosome evolution in species with a rapid generation turnover, as in the Drosophila species.

The Y chromosome of D. melanogaster, in its current state, contains a few protein-coding genes primarily expressed in the testes. With the aid of classical genetic methods, including X-irradiation, chromosome deficiencies, X-Y translocations, P-element insertion mutagenesis, and complementation assays, it has been discovered that the Y chromosome contains at least six distinct loci required for spermatogenesis and male fertility ^{[55][56][57][58][59][60][61][62][63]} (Figure 1a). These six loci encoding the so-called male fertility factors are located both on the long arm (kl-5, kl-3, kl-2, and kl-1) and the short arm of the Y chromosome (ks-1 and ks-2) ^{[57][60][64][65]}. The kl-2, kl-3, and kl-5 genes encode dynein heavy chain proteins that are essential for proper axoneme building in elongating spermatids. Kl-2 is an inner dynein arm heavy chain protein and Kl-3 and Kl-5 are outer dynein arm heavy chain proteins ^[66][67][68]. Note that D. melanogaster has several other dynein heavy chain genes, located on the chromosomes X, 2, and 3 ^[69]. The axoneme is the microtubule-based main part of the sperm flagellum, a specific motile organelle of spermatids and mature sperm. In the motile flagellum, dynein ATPase motor proteins provide sliding motions between adjacent microtubules, which together produce well-ordered movements ^[70]. Deficiency of the kl-3, kl-5 or kl-2 genes leads to loss of the outer dynein structure of the axoneme, and these mutants do not produce motile sperm, resulting in male sterility ^[65][67][71]. kl-2, kl-3, and kl-5 mutant males exhibit clear defects in spermatid morphology and development with the loss of synchronization of their individualization complexes; in addition, they contain short and curled spermatids, nuclei of which are scattered instead of remaining tightly clustered, as in wild-type flies ^[65]. The gene sequences of ks-1, ks-2, and kl-1 had not been identified for a long time, complicating their disruption and studying. However, according to a recent study, the CCY gene located near the telomere of the Y short arm is thought to encode male fertility factor ks-2. RNAi-knockdown of CCY results in short and curled nuclei of elongating spermatids and in male sterility ^[65][72]. In addition, RNAi-knockdown of the WDY gene from the kl-1 locus leads to male sterility, supporting the conclusion that WDY encodes the Kl-1 fertility factor ^[72].

According to publicly available RNA-seq data, most Y-linked genes begin their expression in third-instar larvae and continue to be expressed throughout the pupal stage with the exception of the FDY gene, which is expressed during all developmental stages. Several Y chromosome genes are found to be expressed in imaginal discs, fat bodies, accessory glands, and all of the genes are expressed in the testes ^[65]. In the D. melanogaster testes, generation and transcription of Y-loops take place during primary spermatocyte maturation, coinciding with the data that Y expression peaks in spermatocytes ^[73]. Transcription of the kl-2, kl-3, and kl-5 genes starts in primary spermatocytes and continues during the whole 80–90 h of meiotic G2 phase. Their spliced transcripts are stored in cytoplasmic RNP particles, called kl-granules, in mature spermatocytes along with the ATPase proteins Reptin and Pontin ^[74][75]. These RNP granules segregate during the meiotic divisions and the stored dynein transcripts undergo delayed translation, occurring post-meiotically. Then the dynein proteins are incorporated into the axoneme during the spermatid elongation process ^[75].

Fertility factor genes contain unusually large, megabase-sized introns filled with simple satellite repeats in the cases of kl-5, kl-3, and ks-1 genes ^{[1][64][76][77][78]}. For instance, the kl-3 gene spans at least 4.3 Mb, while its coding sequence contains only approximately 14 kb ^[64][76]. The giant introns comprise more than 99% of the whole kl-3 locus. Transcription from kl-3, kl-5, and ks-1 loci in spermatocytes leads to the appearance of lampbrush-like nucleoplasmic structures named Y-loops A (kl-5), B (kl-3), and C (ks-1) (Figure 1a,b), that are visible with the aid of phase-contrast microscopy, and are analogous to those in amphibian oocytes ^[76]. The kl-5 locus contains four different satellite repeats; the loop-forming site of kl-5 accumulates (AAGAG)n, (AAGAC)n, and (AAGAGAG)n repeats, while the kl-5 non-loop-forming region contains (AATAT)n repeats. Similarly, the ks-1 locus encompasses (AAGAG)n and (AAGAC)n repeats mapping to the loop-forming region in contrast to (AATAAAC)n and (AAGAG)n repeats occupying the non-loop-forming regions. The kl-3 loop is composed of a thinner filament and exhibits a rather diffuse appearance. Only (AATAT)n repeats have been mapped to the kl-3 loop-forming region ^[1].

Y-loop generation reflects the high transcription levels of the underlying genes. Proper transcription of giant genes requires high processivity of RNA polymerase II (Pol II). The presence of long satellite arrays in the introns can lead to the slowing of elongation or frequent premature dissociations of RNA polymerase. Satellite repeats can form high-order DNA, RNA, or DNA–RNA hybrid structures, which may inhibit transcription elongation. Thus, transcription of gigantic intron-containing genes requires precise regulation, and Y-loops contain chromatin associated with a large number of transcripts and regulatory proteins ^[77][79]. Three RNA-binding factors, Blanks, Hephaestus (Heph), and Maca, are found to be enriched specifically in the chromatin of the Y-loops ^[74][80][81]. The corresponding mutations lead to male sterility owing to defects in sperm individualization, similar to the ones observed in males with knockdowns of the kl-5, kl-3, and kl-2 genes ^[74][81]. These proteins are required for transcription or proper processing of the Y-loop gene transcripts. Blanks is found to be located to the Y-loop B (comprising mainly kl-3 introns) and is needed for proper kl-3 mRNA expression ^[74][80]. It has been assumed that Blanks maintains Pol II processivity by binding to the nascent transcripts of kl-3 ^[74]. The Heph protein is found to colocalize with Y-loops A and C. It can regulate the expression of kl-3, kl-5, and ks-1 mRNAs in spermatocytes. Heph may be involved in the processing of kl-5 transcripts, including splicing, or in preventing their premature degradation ^[74]. Maca is essential for kl-2 and kl-3 transcription and proper splicing of kl-3 transcripts, because in maca knockdown testes, the skipping of exon 13 causes an internal deletion in Kl-3 protein ^[81]. Recently described testis-enriched transcription regulators, tPlus3a and tPlus3b, appear to be required for the expression of fertility factors kl-3 and kl-5 ^[82] via an unknown mechanism. Some other RNA-binding proteins are enriched on the Y-loops in the spermatocyte nuclei, such as Pasilla and Boule ^[79][83], Rb97D ^[84], and several proteins encoded by unannotated genes (lup-3, lup-4, dolly-1, and dolly-2) ^[85]. However, their functions in Y-loop transcription await further investigation.

Y-loop generation is a conservative feature across the Drosophila genus, including Drosophila simulans, Drosophila yakuba, Drosophila pseudoobscura, Drosophila littoralis, and Drosophila hydei ^{[86][87][88][89][90]}. In the spermatocytes of D. hydei, clearly cytologically visible Y-loops are found, and early studies have uncovered that their transcription is associated with the huge DNA repeats ^[91][92][93]. Although the functional relevance of the gigantic introns still remains unclear, according to some assumptions long lasting transcription of fertility factor genes (around 80–90 h), due to the presence of the gigantic introns, appears to function as a ‘developmental timer’ for spermatocyte growth and differentiation ^[74][81]. Intron size could also play a critical role in the regulation of gene expression. It has been shown for the Ultrabithorax (Ubx) gene in the early Drosophila embryo that its large size causes abortive Ubx transcription during the syncytial divisions, blocking expression of Ubx protein at the syncytial stage ^[94].

The phenomenon of ‘intron gigantism’ occurs across multiple species, including vertebrates, however, little data are available about its functional significance. Several human neuronal and muscle genes are known to bear giant introns. The best-known largest human gene is dystrophin comprising nearby 0.1% of the whole genome, containing 79 exons and spanning 2.2 Mb, with only 11 kb of coding sequence ^[95][96]. Its gigantic introns, also rich in repetitive DNA sequences, are reminiscent of those of Y-linked Drosophila fertility factors. Dystrophin is located on the p21 region of the X chromosome and codes the causative gene for Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) ^[95][97]. Dystrophin is a major scaffolding component of normal muscle, which links cytoskeletal actin, tubulin, and intermediate filaments to the extracellular matrix, and stabilizes the plasma membrane of striated muscle cells. Loss-of-function mutations of the dystrophin gene trigger instability of the plasma membrane and lead to myofiber loss ^[97][98]. Transcription of dystrophin takes place from several promoters in a tissue-specific manner. Full-length Dystrophin is expressed in all striated skeletal, smooth, and cardiac muscles. Shorter isoforms are expressed in brain and retina cells. In case of frameshift mutations, deficiency of the Dystrophin protein leads to severe DMD disease. In case of in-frame mutations, Dystrophin is expressed as a set of mutated proteins either with missense substitutions or deletions or duplications of its internal part, leading to the weaker BMD disease ^[97]. Exon skipping, with the aid of antisense oligonucleotides to skip the problem exons containing premature stop codon mutations or reading-frameshift mutations, is currently used as an approach for DMD therapy ^[99].

Transcription of these extremely large genes and the processing of their transcripts, including splicing, has a high metabolic cost for cells. The study of genes possessing giant introns using the Drosophila model provides a useful insight into the problems of expression of such genes in humans and the pathologies associated with their improper transcription or splicing. Study of the molecular mechanisms of maintenance, transcription, and processing of Y-loop genes in Drosophila may improve understanding of the origin, selection, and regulation of genes with similar structures in other species.

4. Differential Expression of rDNA Loci of Drosophila Sex Chromosomes

4.1. Nucleolar Dominance as a Widespread Phenomenon

4.2. Y-Based Nucleolar Dominance in D. melanogaster Males

4.3. Non-Random Segregation of Sister Chromatids of Sex Chromosomes in Drosophila

4.4. Differential Expression of rDNA Loci in Human

5. Drosophila Y Chromosome in Studying of piRNA Biogenesis and Functioning of piRNA-Clusters

5.1. Brief Description of the piRNA System

5.2. The Y Chromosome as a Major piRNA-Producing Genomic Region in the Fly Testes

5.3. The Y Chromosome in Other Species as a Source of piRNAs

5.4. The Y Chromosome and TEs

6. Conclusions