Is transcription in sperm stationary or dynamic?

Abstract

Transcriptional activity is repressed due to the packaging of sperm chromatins during spermiogenesis. The detection of numerous transcripts in sperm, however, raises the question whether transcriptional events exist in sperm, which has been the central focus of the recent studies. To summarize the transcriptional activity during spermiogenesis and in sperm, we reviewed the documents on transcript differences during spermiogenesis, in sperm with differential motility, before and after capacitation and cryopreservation. This will lay a theoretical foundation for studying the mechanism(s) of gene expression in sperm, and would be invaluable in making better use of animal sires and developing reproductive control technologies.

The development of haploid spermatids into mature spermatozoa requires a lengthy duration and entails a series of complex physiological changes. During spermiogenesis, the spermatid-specific H2B variants are specifically synthesized and expressed in round spermatids to replace the canonical histones. H2B variants have the capacity to open chromatin and form unstable nucleosomes, which facilitates histone acetylation; In the enlongated spermatids, the Brdt proteins combined with hyperacetylation histone mediate the histones removal, leading to the replacement of histones by transition proteins. Finally, the transition proteins are replaced by the smaller protamines [1,2,3,4]. In this way, the chromatin of sperm is condensed gradually and develops ultimately into a highly concentrated structure [5,6,7,8,9,10,11,12,13]. At the same time, the cytoplasm and ribosome of sperms slowly disappeared, accompanying the gradual differentiation of the other organelles, such as mitochondria and Golgi apparatus. As a result, spermatids become elongated cells and develop into tadpole-like cells with a head and tail. They enter the epididymis for maturation and eventually become sperms capable of movement with the potential for fertilization [6, 14]. This ordered maturation process is completed within a single sperm cell under conditions that exist in testis and epididymis, suggesting that gene transcription and translation play an important role in the regulation of this process. Since condensation of chromosomes occurs in spermatids, many scholars believe that transcription is terminated gradually with the compaction of chromosomal structure, presumably no transcription present in mature sperm [15,16,17,18]. For example, the transcription of few genes was detected in post meiosis phase in Drosophila [19], and the transcription was even undetected in late spermatids in mouse [20]. However, an increasing number of studies showed that sperm carry thousands of different types of RNA, including messenger RNAs (mRNA), microRNAs (miRNA), interference RNAs (IRNA), antisense RNAs, etc. [8, 11, 20,21,22,23,24,25,26,27,28,29,30]. In fact, more than 4,000 kinds of mRNAs were found in the studies of human sperm [11, 20, 31]. Due to the belief that gene transcription is silenced in sperm, the large quantity of RNA that nevertheless still remains is therefore hypothesized to exist as relics of spermatogenesis [11, 32]. Before terminating of nuclear transcription, the various mRNAs needed during the stages of spermiogenesis are transcribed in advance and retained for a long period of time; the mRNAs are then translated into proteins to ensure that all functions subsequent to nuclear transcription are normal and continuing [33,34,35]. Concerning that this hypothesis cannot explain the fact that a large number of rRNAs in the cytoplasm are removed or degraded, there are certainly different types of mRNAs left behind. In addition, studies also showed that the histones are not completely removed from nucleosomes in ejaculate spermatozoa, and they contributed to sperm chromatin approximately accounting for 1% in mouse [36], 15% in human [37], and 50% in some marsupial species [38]. As a result, some chromosomal regions of sperm manifest slacker conformations for the retained histones [17, 39], which may allow transcription factors to bind to specific gene sequences, providing transcriptional potential [35, 40,41,42]. In addition, a reverse transcriptase activity was observed in murine epididymal spermatozoa [43]. Here are the questions: are sperm RNAs the remnants from spermatogenesis before the end of nuclear transcription, or is there timely expression from sperm chromosomes; or do both occur? In order to reveal the nature of sperm maturation, improve the reproduction capability, and realize male contraception, the study on this topic is becoming more and more important.

However, the transcript number in sperm is relatively low [44], and all types of the germ line cells mix together. It is difficult to capture cell samples and obtain enough RNA samples for exploring the dynamics of sperm transcription. Currently, the combination of the technology of frozen sections and laser capture microdissection (LCM) overcomes the difficulty of sampling spermatids from different developmental stages during spermiogenesis [45, 46]. In addition, RNA amplification and RNA-Seq are now widely applied. The development of all these techniques is expecting to promote the discovery of the theme.

The transcripts in spermatids vary during post-meiotic

After meiosis, there are some morphological changes, including nuclear shaping and chromatin compaction as well as major cytoplasmic transformations in sperm [47, 48]. These post-meiotic events are considered to be driven by translation, as transcripts are considered originating from primary spermatocytes and stored in spermatids and translated during elongation. However, transcripts were detected in round spermatids [49] and elongated spermatids [50], and transcription of ram sperm chromatin was also examined by two electron microscopic techniques [51]. In 2006, Welch et al. analyzed the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase gene (Gapds) in rat spermatogenic cells at different maturational time-periods using northern blot. They detected Gapds mRNA in round and condensing spermatids but not in primary spermatocytes [52], and demonstrated that the spermatogenetic cell-specific Gapds gene is inactivated in primary spermatocytes, whereas is expressed in the postmeiotic phase of spermatogenesis, and number of Gapds transcripts in condensing spermatids was significantly greater than in round spermatids. The detection of 24 comet and cup genes’ transcripts during Drosophila spermatogenesis and spermiogenesis using in situ hybridization showed that the transcript number for hale-bopp (hale), schumacher-levy (schuy), davis-cup (d-cup), presidents-cup (p-cup), tetleys-cup (t-cup), flyer-cup (f-cup), sungrazer (sunz), and other genes in elongated spermatids was significantly higher than that of in round spermatids. In addition, the transcript number for these genes during the transformation of histones to protamine including complete replacement of histones by protamine shows a significant upward trend, which was proved by Q-RT-PCR [50]. According to post-meiotic transcription of these genes, authors drew a conclusion that transcription in Drosophila stops in late primary spermatocytes, then is reactivated by two pathways for a few loci just before histone-to-transition protein-to-protamine chromatin remodeling in spermiogenesis. Moreover, a surprisingly strong 5-bromouridine (BrU) signal was observed near spermatid nuclei in developing spermatid bundles during postmeiosis, and the BrU signal was reduced in the presence of actinomycin D, a general inhibitor of RNA synthesis [53]. They implied that the BrU signal in spermatids was dependent on RNA synthesis. Study showed that there are two categories of post-meiotic transcriptional regulation: methylation and trans-acting factors that bind to the TATA-box, the CRE-box, or other specific DNA sequence in the promoter region of nucleoproteins [49]. Since these genes are trancriptionally active only before the chromatin remodeling, how will the transriptional activity be after the histones are replaced by the protamines? It is still a highly debatable issue.

The transcripts in sperm vary with different sperm motilities

Motility is necessary for sperms to be able to penetrate cervical mucus, enter the fallopian tube, and eventually bind to the oocyte. Since the motility of sperms may vary among different animals and even among different sperms from the same sire, here we focus upon transcript variation among sperms with different motilities. The androgens/estrogens balance is essential for normal sexual development and reproduction in mammals. The P450 aromatase (P450arom) encoded by cyp19 regulates the balance of androgens and estrogens by catalyzing the demethylation of androgen to be oxidated to estrogen [54, 55]. Recently, the P450arom transcripts were found to be significantly different between immotile and motile sperms. Compared with motile sperm fraction from the same sample, a 28–30% decrease of the amount of P450arom mRNA is observed in immotile sperms [56]. While for the genes of the protamines PRM1, the opposite was observed. Lambard et al. (2004) found the number of PRM1 transcripts in low-motility sperms was significantly higher than that in high-motility sperms [57]. On the contrary, Ganguly et al. (2013) found that the amount of PRM1 mRNA in normal-motility sperms was significantly higher than in low-motility sperms [58]. It appears that the quantity of PRM1 transcripts varies according to the sperm motility, and further evidence is needed. Evaluation of endothelial nitric oxide synthase (eNOS) gene and neuronal nitric oxide synthase (nNOS) gene showed that the two transcripts were undetectable in most of the high-motility sperms, and only detected in low-motility sperm samples [57]. The high levels of eNOS and nNOS transcripts in low motile sperms may result in the excessive production of NO, which is responsible for the inhibition of sperm motility [59]. Genes of sperm cation channel-like protein family play important roles in different aspects of mammalian sperm functions, such as sperm motility, capacitation and the acrosome reaction [60, 61]. Their transcripts’ quantity is different in sperms with different motility. For instance, the transcript level of CatSper2 and CatSper3 in high-motility sperms was significantly higher than that of in low-motility sperms [62]. Jing et al. revealed a positive correlation between CatSper1 transcript level and sperm motility [63]. Additionally, Chen et al. unveiled that the number of expressed nuclear factor erythroid 2-related factor 2 (NFR2) gene in low-motility sperms was significantly lower than in high-motility sperms [64].

The transcript number of genes in sperm appears to vary with different motilities, attributing to an increase of the transcriptional activity, a decrease at the translational level or a longer half-life of the RNAs [56, 65]. Nevertheless for the same amount of RNA analyzed, the level of specific P450arom transcript was significantly lower in the immotile sperm cells, as also reported for the PAF-receptor mRNA [66]. A recent study showed that the transcript quantity of the mitochondrial NADH dehydrogenase 2 (MT-ND2) gene in asthenospermic sperms was significantly lower than in normal-motility sperms, as was the transcript number of three genes annexin A2 (ANXA2), bromodomain containing 2 (BRD2), and ornithine decarboxylase antizyme 3 (OAZ35). Among them, the transcripts of ANXA2 and BRD2 were positively correlated with sperm motility [67]. The quantity of transcripts is different in sperms with different motility, and this difference leads to a series of discussion questioning the presence of sperm transcriptional activity. Detection of low level transcription in sperms, especially under certain conditions such as capacitation, and acrosome reaction, has been documented [52, 68]. Further verification is needed to support the idea.

The transcripts in sperm vary with capacitation

Unless they undergo capacitation, mammalian epididymal and ejaculated sperms do not have the ability to fertilize the oocyte in vitro [69, 70]. It has been confirmed that sperm proteins change after capacitation [21, 71]. Lambard et al. (2004) found that protamine transcripts did not significantly change, but the c-myc transcripts partially or completely disappeared in the sperm of healthy humans four hours after capacitation. Lee et al. (2011) analyzed the transcripts of Myc, CYP19A1 encoding aromatase, domain-containing protein 2 (ADAM2), PRM1 and PRM2 in pig sperms before and after capacitation by RT-PCR and quantitative real-time PCR. Their results showed that the transcriptional level of PRM1 and PRM2 did not significantly change, but MYC, CYP19A1, and ADAM2 was significantly down-regulated after capacitation [72]. The decrease of some transcripts after capacitation might result from the increase of the translational activities during capacitation for more protein synthesis [54, 73]. Transcriptional activities in the head and midpiece regions of sperm during capacitation had been detected, although the studies on transcript increase had not yet been reported [68]. It needs to be further verified whether the transcriptional activity increase or not after capacitation.

The transcripts in sperm vary with cryopreservation

Semen cryopreservation promotes the application of artificial insemination (AI) in livestock breeding, and draws more attention to the impacts of cryopreservation on sperm transcripts. Ostermeier et al. (2005) tested the expression of the expressed sequence tags (ESTs) from human sperm samples exposed to different freezing-thawing cycles [74]. The authors found that the number of ESTs in fresh semen was highest and there were 59 more ESTs in sperms treated with one vs three freezing-thawing cycles. Garcia-Herrero et al. detected the transcripts in fresh and frozen sperms used for intracytoplasmic sperm injection (ICSI) and analyzed the differential expression between sperms that resulted in pregnancy and those that didn’t (2011). Transcripts of 19,229 genes were detected in fresh semen, while 18,095 were found in frozen semen. The transcript difference was also found in fresh sperms between pregnancy and nonpregnancy groups, while no difference was detected in frozen spermatozoa between pregnancy and nonpregnancy groups [75]. In addition, Valcarce et al. (2013) found that the transcript number in sperms after freezing treatment was significantly reduced [76]. Therefore, frozen treatment significantly decreased the number of transcripts in sperms, and the more frequent the freezing-thawing treatments, the fewer the number of transcripts. Among these transcriptional variations, different trends for different genes were observed. The transcript number of an RNA-binding protein gene CIRBP in bovine frozen sperm was reduced, while the transcript levels of genes encoding cold shock protein A (CspA), heat shock protein 60 (HSP60), and heat shock protein 10 (HSP10) were increased after freezing and thawing [77]. These two trends were also detected in Chen’s study (2015). Transcripts of 16 genes were significantly increased and transcripts of 3 other genes were significantly reduced after cryopreservation. The up-regulation of PRKCE and unknown geneR1G7 after cryopreservation may be related to anti-oxidation and strengthening of the acrosome reaction [78]. Recently, a study on boar spermatozoa found that the expression level of 3 microRNA in cryopreserved spermatozoa are higher than in fresh ejaculate [79]. This variation of transcript quantity between frozen and fresh sperm may be induced by the freezing-thawing treatment, which maybe affect mRNA–protein interaction and make mRNA more susceptible to degradation [76]. The increase of transcript may be due to the freezing stress which led to the transcriptional activity increasing [80].

Discussion

Sperm constitutes the only vector that can convey and perpetuate life for male. As a single haploid cell, sperms perform all of their vital processes within the female reproductive tract, including capacitation, movement, and recognition and binding with the ovum.

Sperm can adapt to the external environment so as to complete this series of crucial events requisite for life, and a series of transcriptional and translational events may thereby play an important role. Transcripts vary with spermiogenesis and capacitation state, and they also vary with differential sperm motilities, the freezing-thawing cycle (Table 1), and different sex chromosomes the sperm carries [80]. However, chromatin structure is compacted in sperms, it deserves further study to investigate whether it has the potential for transcription to successfully regulate the viability of this single reproductive cell.

Table 1. Different opinions about transcriptional activity in mature sperm.

Opinions	References	Species	Method
Post-meiotic transcription	Welch et al., 2006	Rat	Northern blotting
	Barreau et al., 2008	Drosophila	Situ hybridization
The transcripts in sperm vary with different sperm motilities	Lambard et al., 2003	Human	RT-PCR
	Lambard et al., 2004	Human	RT-PCR
	Li et al., 2007	Mouse and human	RT-PCR
	Jodar et al., 2012	Human	qPCR
	Chen et al., 2012	Human	qPCR
	Ganguly et al., 2013	Bull	qPCR
The transcripts in sperm vary with capacitation	Lambard et al., 2004	Human	RT-PCR
	Lee et al., 2011	Human	Microarrays
The transcripts in sperm vary with cryopreservation	Ostermeier et al., 2005	Human	Microarray
	García-Herrero et al., 2011	Human	Microarray
	Valcarce et al., 2013	Human	qPCR
	Chen et al., 2015	Bull	Microarray

There are different RNA populations in mature sperms from fertile and infertile men, and some highly associated with sperm motility, capacitation, and other parameters [57, 59]. Another important point is that some sperm RNAs are present in zygotes and early embryos, and regulate epigenetic events affecting embryonic development or function as a regulator participating in cell signaling processes during development of the zygote and embryo [81]. Thus, the study of sperm transcript may have profound clinical implications in the diagnosis of male infertility as well as in the practice of assisted reproductive technologies. Are the sperm RNAs which play important roles the remnant of spermatogenesis or the result of sperm transcription? It is not clear at present and needs to be further studied.