Discussion
Based on the phenotypic variation of sperm in tree sparrow lived in BY
which has been detected in our previous researches, we uncover a
dramatically expanded PIM gene family may play an important role
in shaping the phenotypic change of sperm under environmental pollution.
As the only gene overlapped between the PSGs and DEGs, PIM1 is
identified as candidate gene for sperm evolution and targeted as the
focus of our research. Although there is no documented example ofPIM1 enables rapid evolution of sperm, the genetic variation and
differentially expression of cAMP-dependent protein kinase (PKA ),
another serine/threonine protein kinase which have high sequence
homology with PIM1 (Jacobs et al., 2005), is associated with
rapid evolution of sperm in deer mouse (Peromyscus maniculatus )
has been reported (Fisher et al., 2016).
As soon as our focus are narrowed down, the unusual and large-scale
expansion of PIM gene family mainly caused by the duplication ofPIM1 are noticed in the genome of tree sparrow. 449 PIM1genes were predicted from the tree sparrow genome assembly and 142 of
them are complete for the kinase domain. Gene duplications provide a
source of genetic material for mutation, drift and selection to act
upon, making new evolutionary opportunities for rapid adaptation (Crow
& Wagner 2006; Hu et al., 2022; Magadum et al., 2013). Duplicate genes
have been proven to be important targets of positive selection in
Arabidopsis (Moore & Purugganan, 2003). As a result, the large number
of duplicate PIM1 gene copies may provide raw materials that
facilitate rapid evolution of sperm when selective pressures shift in
BY, which is demonstrated from the side by the fact that multiplePIM1 duplicates appear repeatedly in outlier regions.
Our analysis also finds that both tandem duplication and segmental
duplication events are involved in the expansion of PIM gene
family, and the segmental duplications cause simultaneous expansion of 6
other gene families and LTR retrotransposons adjacent to PIM on
chromosomes. The adjacent chromosomal distribution and simultaneously
expansion suggest that the 7 families co-evolved in genome, and it is
noteworthy that the expansion of these 7 gene family is so dramatical
that make it may become a strong driving force of the evolution of tree
sparrow genome.
Among the 7 expanded gene families in tree sparrow, the duplications of
the most two prolifically expanded gene familiesC2H2ZNF and OR are
widespread and prevalent in other species (Freitag et al., 1998;
Seetharam & Stuart 2013) relative to the other 5 gene families. The
duplication of PIM1 has only been reported in zebra finch (Kong
et al., 2010) whereas PIM1 is believed to be single-copy and
conserved in other amniotic genomes, even in chicken. Our analysis ofPIM1 duplication events in other avian genome indicate thatPIM1 expansion is not peculiar to passerine but arose in several
lineages during the evolution of bird. Fixation of duplicate genes
occurs when natural selection favors function or expression of
duplicates (Cardoso-Moreira et al., 2016; Chain et al., 2011). Although
the protein activity of duplicate PIM1 has not confirmed yet, compared
with the species with single-copy PIM1 , the retention of
duplicates in genome and the increased gene dosage may suggest a more
important and beneficial function of PIM1 in the evolution and
adaptation of the species with PIM1 expansion. At the same time,
there still much less PIM1 duplicates are found in any other
avian genome than in tree sparrow which may due to the further expansion
of PIM1 in tree sparrow than other species or the incomplete
genome assemblies.
The expression pattern of PIM1 seems to be different in different
species. PIM1 has been reported to be expressed mainly in testis
and hematopoietic tissues including thymus, spleen, bone marrow and
fetal liver in both mouse and man (Bachmann & Möröy, 2005; Eichmann et
al., 2000) when PIM1 expressed in the brain and testis of zebra
finch (Kong et al., 2010). Based on the current evidence, it seems that
testis is only tissue in which PIM1 of mammal and passerine both
expressed, which indicates that PIM1 may play an important role
in male reproduction. But besides a few early researches implyPIM1 is implicated in the normal development of male germ cells
(Sorrentino et al., 1988; Wingett et al., 1992), the functions related
to male reproduction of PIM1 are fleetingly addressed in studies
and people pay more attention to its role in cancer (Narlik-Grassow et
al., 2014) and brain function of songbird (Kong et al., 2010). A large
number of duplicates and the testis-specific expression pattern, as
revealed by our analysis, as well as the involvement of mammalPIM1 in normal germ cell maturation published before (Sorrentino
et al., 1988; Wingett et al., 1992) may indicate PIM1 play a
strong and essential role in spermatogenesis of tree sparrow. We thus
consider the role of PIM1 in male reproduction, an indispensable
and inseparable part of PIM1 function, should never be neglected
not only in the species with multiple PIM1 but in species with
conserved single-copy.
Higher expression levels of PIM1 , wholly or individually, are
detected in testis sampled from BY tree sparrow. Interestingly, the
expression level of PIM3 , a member of PIM family as well
as PIM1 but without duplication and testis-specific expression
pattern, shows no difference between BY and LJX. The elevated expression
level may be the consequence of shifts in selective pressures on
duplicate PIM1 . Furthermore, we test if the elevated expression
level of PIM1 in polluted site is due to larger scale of
duplication and higher copy numbers of PIM1 . However, the results
show that there is no increase in copy number of PIM1 in BY
population. Therefore, the molecular mechanism of change in expression
level of PIM1 still need to be further researched in the future.
When we take into account the link between transcriptional changes to
sperm phenotypic effects, the higher expression levels of PIM1seem to be consistent with our earlier findings of longer and faster
swimming sperm in BY (Yang et al., 2020a, 2020b). No research is found
to explore the relationship between expression level of PIM1 and
sperm traits variation, nonetheless, several studies have proven thatPIM1 can accelerate cancer cell motility and overexpression ofPIM1 result in increased cancer cell motility (Mou et al., 2016;
Santio et al., 2020; Tanaka et al., 2009). PIM1 kinase normally
accelerates cell motility through phosphorylating and activating target
proteins. We thus speculate that PIM1 also accelerate sperm motility
through phosphorylating downstream proteins in tree sparrow. However,
there are so many proteins that can be phosphorylated by PIM1. In
prostate cancer cells, PIM1 can accelerate cell motility through
phosphorylating actin capping proteins (Sanito et al., 2020). PIM1
accelerate human oral squamous cell carcinoma cell motility through
activating Ras-related C3 botulinum toxin substrate 1 (RAC1) instead
(Tanaka et al., 2009). Therefore, it is hard to confirm the target
protein and signaling pathway of PIM1 in tree sparrow. We attempt to use
PIM1 inhibitor to verify the molecular function and preliminary explore
the downstream protein of PIM1 kinase. However, there is no observed
sperm trait change are found in the inhibitor treatment group. The
unchanged sperm trait may due to the limitations of our inhibition
experiment. We cannot ensure whether the inhibitor can pass through the
blood-testis barrier and reach the target tissue, or whether the mammal
inhibitor can work equally on tree sparrow.
In conclusion, our work utilizes population genomic and transcriptomic
studies, exploring the genetic underpinning of phenotypic variation of
sperm in tree sparrow lived in BY which is polluted by heavy metal. On
the one hand, our results find PIM1 , a single-copy gene in most
of amniotic genomes, remarkably expanded in tree sparrow and other avian
species of three lineages including Passeriformes, Psittaciformes and
Strigiformes. Duplication of PIM1 may be an important force
driving speciation and phenotypic diversity in avian evolution. On the
other hand, while the molecular functions and pathways of the expandedPIM1 remain to be defined by experimental verification, the
signatures of selection and upregulated gene expression in polluted
sites, indicating the expanded PIM1 may be important sources for
rapid evolution of sperm in tree sparrow following the environmental
change.