Andrology-Open Access
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Recent advances in the human male gamete: focus on testicular varicocele

Adele Vivacqua^1,2, Giuseppina Peluso³, Marco Serrao⁴, Lidia Urlandini¹, Mattia Saporito¹, Helena Adamo², Vittoria Rago¹ and Saveria Aquila^{1,2* *}Correspondence: Saveria Aquila, Department of Pharmacy and Science of Health and Nutrition, University of Calabria, Cosenza, Italy, Email:

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Introduction

To date, it is known that the morphologically mature male gamete presents unique features: it is a highly differentiated cell that displays extreme polarization of cellular composition and function. For example, the sperm head has evolved to interact with the egg's extracellular matrix and to transport paternal genetic material, whereas the sperm flagellum provides motility. Sperm possesses little cytoplasm and therefore has a reduced ability to translocate substrates from one region to another. It is considered transcriptionally inactive due to DNA hypercondensation by protamine and therefore cannot make new proteins in response to changing needs. The human sperm passes through two states of development: the first in the male genital tract, where it acquires morpho-anatomical maturation; the second in the female genital tract, where it acquires functional maturation during the capacitation process, which prepares the gamete for the acrosome reaction. In recent years, new and intriguing aspects have emerged from this cell, including autocrine regulation of its functions by sperm-derived factors, the presence of different types of mRNAs, and sperm ability to translate through mitochondrial ribosome-type, both from mRNA carried from early spermatogenesis or from fresh RNA. From our studies, it emerges that studying the anatomy of human sperm at the molecular level is crucial, as new sites of expression of molecules traditionally confined to specialized cells, such as insulin, leptin, and estrogens, possibly operate through a short autocrine loop [1-3]. The different types of mRNAs have been associated with sperm translation. Altogether, these studies open new chapters in andrology, since sperm that appear morphologically normal may not always be able to fertilize an oocyte.

From this, it may be disclosed that sperm can be considered a mobile endocrine unit independent from systemic hormonal regulation [1-3]. This is likely due to the peculiar characteristics of sperm, since at any given time it is free from supervision of the organism that produced it: the spermatozoon leaves the testis to meet the unknown, moving through the female genital tract in the host body of the opposite gender, requiring autonomy. Our studies on autocrine regulation of various sperm activities, including metabolism, define a new endocrine and metabolic sphere for human sperm [1-3].

Some years ago, a population of mRNAs was observed in sperm, some of which are delivered into the oocyte together with the paternal DNA. It has been shown that the presence of some of these mRNAs is essential in early embryonic development, since their silencing prevents progression [4-7]. The mRNAs present in sperm include thousands of distinct species [5]. Understanding the messages carried by the male gamete as mRNAs may lead to a redefinition of paternal and maternal roles in fertilization and embryonic development [7, 8]. The oocyte recognizes the sperm as a potential harmful intruder. Our recent study showed that sperm can defend itself in physiological and pathological conditions through a sperm fight/flight response, producing cortisol aimed at assaulting the immune system [9].

The transmission of parasites’ genetic paternal derivation (retrotransposons and endogenous retroviruses) is a mechanism that uses both PIWI-interacting RNA (piRNAs) and microRNA (miRNAs) in the oocyte’s plasma [10]. However, these interactions at the mRNA level can be associated with chromatin modification of histones that scan the paternal genome input to assess genetic compatibility. Sperm that passes recognition for cytoplasmic and genetic compatibility can proceed to syngamy [7]. Pronuclei may form regardless of genetic compatibility, but failure to pass leads to a failure in activation of the embryonic genome [7].

Until recently, sperm was thought to act only as a cargo to transport paternal genome to the oocyte. However, other components of the spermatozoon essential for normal syngamy in all investigated mammalian orders (except some rodents) have come to light. These include an oocyte activating factor (a member of the phospholipase group), an essential component of the zygote’s microtubule-organizing center (the spermatozoa centrosome), and mitochondria [11].

Over the past years, reports have described the presence of various mRNAs in male gametes of many species, including gymnosperms and angiosperms [12, 13]. These studies have renewed interest in RNA carriage by spermatozoa because it is an anomalous component of this apparently ‘quiescent’ specialized cell. Nuclear gene expression in mature spermatozoa is progressively shut down during spermiogenesis (the haploid phase of spermatogenesis) to allow substitution of histones by the highly charged and smaller protamines, facilitating further compaction of the haploid genome [14]. Although sperm mRNAs have been debated for over 40 years, it is now generally accepted that spermatozoa carry a large population of mRNAs, antisense RNAs, and miRNAs into the oocyte together with paternal DNA [4]. However, their functional significance remains debated. This reflects that spermatozoa have a compacted nucleus and are transcriptionally inactive. Nevertheless, spermatozoa transmit mRNAs to the oocyte at fertilization as part of the multilayered paternal contribution, which also provides essential organelles (centriole and mitochondria in humans and primates) and male-specific proteomic components (PLCz, PT32, STAT4, ETS) [15].

The localization of mRNAs in sperm nuclei has been reported, and their compartmentalization within euchromatic and heterochromatic regions of the genome has been described [10]. The retention of mRNAs and their association with the nuclear matrix is particularly intriguing given the differential packaging of spermatozoal DNA. During spermiogenesis, most histones are progressively replaced by the smaller, basic protamines, compacting the nucleus to 1/13^th that of the oocyte [14]. During this period, transcription ceases, and spermatids rely only on stored mRNAs to support protein synthesis [16]. However, protamine repackaging is not complete in many species, as in human spermatozoa, which retain approximately 15% of their DNA as histone-bound and thus likely available for transcription [16].

Different outcomes have been proposed for RNA populations mRNA, antisense RNA, and microRNA retained in mature sperm:

1. Antisense and microRNAs may epigenetically modify and modulate early embryonic gene expression.
2. Sperm mRNAs may also have a structural role. For example, nuclear RNA may target chromosome regions remaining histone-bound.
3. mRNA located in the midpiece may be translated under certain conditions, for example, during capacitation.
4. The vast majority of DNA within the head is packaged by highly charged protamines [14]. However, a small proportion of the genome is packaged by histone-containing nucleosomes. It is proposed that these segments are marked with paternally derived mRNAs [7, 17].

Literature Review

Translation ability

It was shown that the male gametes are capable of de novo translation by ribosomes like those mitochondrial during capacitation [18-22]. The mechanism by which the nuclear-encoded mRNA transcripts are translated by ribosomes mitochondrial presently is unclear. Two possible mechanisms have been hypothesized: The first provides that mRNA transcripts can be translated by CP-sensitive ribosomes (Chloramphenicol) that are located outside of the mitochondria but anchored to its outer surface; alternatively, the mRNA nuclear power could be imported into the mitochondria to be translated by CP-sensitive ribosomes within the mitochondria [21,22]. The import of tRNA and rRNA in the mitochondria has been described in several species, import of mRNAs in mammalian mitochondria is unprecedented [7]. One way to ensure the transport of a nuclear protein to another cellular organelle, is to transport the mRNAs that encodes the protein to the ribosome which is located into the correct subcellular compartment. The mitochondrial precursors of the proteins carry the targeting sequence of amino terminal in such a way as to ensure their passage in the mitochondria [18,21,22]. The mRNA transcripts encoding mitochondrial proteins do not meet the mitochondria to mere chance, they use a special mechanism for fast track of mRNA, which is the first step of the sorting of mitochondrial proteins [18]. The second hypothesis suggests that protein translation takes place within the mitochondria. Studies demonstrated that the RNA translation in mature sperm happens in the mitochondria and seek to further localize the site of mRNA translation [21-22]. In mammalian sperm nuclear mRNA is translated by ribosomes like mitochondria in the cytoplasm and mitochondria, later translated proteins are activated [21-22].

Imprinting

Mounting evidence established that antisense RNAs play a critical role in unchanging silenced chromatin domains. There is the possibility, that the recently discovered antisense spermatozoa mRNAs could provide an epigenetic mark that is necessary for establishing and/or maintaining paternal imprints [6,17].

Under certain conditions, some cytoplasmic mRNAs are apparently translated de novo, possibly in mitochondrial polysomes. When this translation is prevented, sperm maturational events associated with capacitation are not observed [21-22]. This would suggest that the production of proteins from spermatozoal mRNAs is necessary for the maintenance of male fertility. In addition to structural roles, it has been suggested that in common with oocyte mRNA stores, spermatozoa transcripts may be functionally important to the zygote to promote post-fertilization development. Recent evidence would also suggest that paternal RNAs can provide epigenetic marks to the developing embryo that influence the phenotype of the offspring. Embryonic development, pattern formation, embryogenesis, and tissue organization [6,7,17].

These terms all refer to the processes by which an embryo develops its specific spatial arrangement of cells and tissues. Combining the identification of spermatozoa mRNAs within the midpiece and the dependence of mRNAs distribution by the paternally derived centrosome, it may be speculated that paternal transcripts play a key role in spatial patterning of the developing zygote [7].

The oocyte recognizes the sperm as a potential harmful intruder and then it requires a careful control before sperm-derived factors can be accepted for the syngamy. From the oocyte’s perspective, the incoming sperm poses a significant challenge. The sperm is a “foreign” body that may carry with its undesirable factors into the egg [7].

The activation of the embryonic genome requires interactions of complementary mRNAs from oocyte and sperm which scan the paternal genome to assess genetic compatibility. The sperm that passes the recognition for the cytoplasmic and genetic compatibility can proceed to syngamy. A pronuclei regardless the check in for genetic compatibility may be formed but this will lead to a failure of the embryonic genome activation. Many failures of fertilization or developmental failure (through both natural and assisted conception) could be attributed to the sperm being unable to pass the checks necessary for successful syngamy and embryonic genome activation [7].

De-novo translation of spermatozoa mRNAs has been convincingly demonstrated in human and bovine spermatozoa as visualized by autoradiography and fluorescence microscopy, by the incorporation of labeled amino acids into polypeptides during sperm capacitation [21,22]. Surprisingly, mitochondrial (D-Chloramphenicol (CP), tetracycline and gentamycin) but not cytoplasmic translation inhibitors (cycloheximide) prevented translation. Inhibition of protein translation significantly reduced sperm motility, capacitation, and in vitro fertilization rate. Thus, contrary to the accepted dogma, nuclear genes are expressed as proteins in sperm during their residence in the female reproductive tract until fertilization. It was shown that the male gametes are capable of de novo translation by 55S ribosomes like those mitochondrial during capacitation [21,22].

The spermatozoon’s mitochondrial transcription-translational machinery, however, is active; hence the work demonstrating translation de novo essentially confirms the conclusions drawn 50 years ago in this regard [21,22]. In general, the prevailing consensus is that although mRNA and protein synthesis does occur in mature spermatozoa, they are confined to the cells’ mitochondria. What is entirely novel about the most recent work is the evidence that polynomial complexes containing mitochondrial ribosomes may support the translation of nuclear-encoded cytoplasmic mRNAs. The mechanism by which the nuclear-encoded mRNA transcripts are translated by ribosomes mitochondrial presently is unclear. It is known that 16S and 12S ribosomal RNAs are present in the nucleus of mouse and human spermatozoa [21,22].

With respect to the role(s) of spermatozoa RNA, it has been discussed the evidence. This suggests that it is not residual from spermatogenesis. Possible functions include de-novo translational replacement of degraded proteins (now demonstrated), structural (repackaging of chromatin), post-fertilization and epigenetic. Perhaps the most exciting development to date is the inference of naturally occurring spermatozoa RNA-mediated epigenetic effects on the zygote, but detecting these effects remains a challenge for future research [6,7]. Certainly, the renewed interest in the male gamete is both welcoming and timely.

Active spermatozoa

Although it is now widely accepted that under normal conditions, spermatozoa are transcriptionally silent, recent evidence indicates that the spermatozoa nucleus is itself more dynamic than was originally considered. In addition to the new evidence for active translation of stored mRNAs, the transcription of fresh RNA, was hypothesized because (i) spermatozoa contain RNA polymerase (ii) and abundant transcription factors are present [23]. It is likely that spermatozoa synthesize new proteins needed for capacitation to replace proteins that have degraded. Protein translation is essential for sperm functions that contribute to fertilization, such as motility, actin polymerization, and the acrosome reaction. Thus, the ability of spermatozoa to synthesize proteins, including nuclear-encoded proteins, by the 55 S ribosomal machinery is critical for the final maturation step leading to successful fertilization [21-22].

Steroids

Spermatogenesis is one of the most important targets of steroids and it was recently demonstrated their ability to influence the activities of the male gamete [1-3,24]. Nevertheless, in the literature controversial data are reported, the main problem for andrologists is to accept the presence of transcription factors, such as the steroid receptors, in a cell that appears silent from a transcriptional point of view [25,26]. The most of studies have supported the hypothesis of membrane receptors for steroids by which they can induce rapid effects. It is now clearly knowing that the classical steroid receptors act also by inducing rapid effects [27].

Estrogen Receptors (ERs): the effects of estrogens are mediated by two distinct nuclear receptors, ERα and ERβ. The receptors are encoded by two different genes and are expressed in germ cells. Besides the genomic effect mediated by the two nuclear receptors, estrogens also because non-genomic effects mediated through membrane receptors. Estrogen stimulates the phosphorylation of proteins involved in the cascade of Phosphatidylinositol-3-OH Kinase (PK3)/Akt and stimulates ERK1/2, which are involved in sperm function. The presence of ERα has been reported only in the midpiece of sperm, ERβ exhibit wider distribution patterns along the sperm tail, extending through the midpiece and the principal piece of flagellum. The sperm head appears to be totally devoid of both these proteins [28]. The Transmission Electron Microscopy (TEM) with immunogold analysis, confirmed the abovementioned location of these steroids’ receptors [29].

Androgen receptor: androgens are steroid hormones important for the onset and maintenance of spermatogenesis as well as the development of secondary sexual characteristics. They act by genomic and non-genomic effects, mediated by nuclear receptors [30]. By immunofluorescence and confocal microscopy, have been localized ARs in the midpiece and in the mitochondria of human ejaculated sperm [30]. Furthermore, the classical AR-B and AR-A isoforms were shown to be present in sperm by western blot and TEM mostly within the head and in the midpiece [30].

Progesterone receptor: progesterone, a female hormone, is also involved in male reproduction. The physiological responses of progesterone are mediated by two types of nuclear receptors, the PR-A and PR-B receptor. These two classic nuclear receptors are encoded by a single gene. Similarly, to the estrogens and androgens, progesterone also binds to the surface of the sperm through specific receptors located on the membrane. Recently, the expression of the conventional intracellular PRs has been demonstrated, and their functional role has been related to capacitation, p-Akt and p60c-src activities, acrosome reaction, lipid, and glucose metabolism. TEM with immunogold examination localized the PRs not only to membranous compartments but also in the entire sperm body as component of the nucleus, the midpiece and the flagellum [31].

Testicular varicocele

Although varicocele is the most common cause of secondary male infertility, pathogenesis and its effects are still topics of investigation. The etiology and physiopathology of varicocele remain unclear and the mechanisms by which varicocele causes testicular dysfunction and infertility are not completely known. The effects of varicocele occur at different levels [32,33] (Figure 1).

Figure 1: Schematic representation of varicocele influence on male fertility.

Effect on spermatogenesis: Experimental data from clinical and animal models show that rising temperatures because of venous reflux, damage spermatogenesis [34]. In addition, the varicocele causes endocrine imbalance and hyperthermia in the testicles, oxidative stress, apoptosis and dysfunction of the epididymis [35].

Effect on seminal fluid: Many studies have tried to highlight the seminal indicators of the disease, but even here there are controversial data. In fact, the condition of the varicocele may be associated with several seminal paintings that veer from normozoospemia to moderate oligoastenoteratozoospermia or azoospermia [36].

Effect on gamete: The impact of the disease on the functionality of the gametes is still debated by scientists. Some studies have shown a significant association between varicocele and low quality of gametes; however, few studies have focused on the effect of the disease on the functions and structure of the human male gamete [32].

The varicocele induces an altered morphology with a prevalence of pointed heads, in fact, semen quality in varicocele patients is characterized by tapered sperm cells, furthermore, studied the effects of the disease by TEM showing a combination of generic characteristics as non-condensed nuclei, malformed acrosome, big waste cytoplasmic, not tightly assembled mitochondria, rolled axonemes [37]. Sperm ultra-morphology as a pathophysiological indicator suggested that varicocele may induce deleterious alterations in early spermatid head differentiation, causing sperm acrosome and nucleus malformations. In this context, our studies have shown that this pathology induces damage in the male gamete at the molecular level which includes a reduction of some steroid receptors, going beyond the morphological abnormalities described to date. Our research group has shown firstly the presence of classical receptors for steroids in human ejaculated male gamete at both the protein and mRNA [1-3]. Their function was related to motility, survival, capacitation, and acrosome reaction. To evaluate the possible involvement of ERs, PR-A / PR-B and ARA / ARB steroid receptors in the pathophysiology of varicocele, we investigated their expression and ultrastructural localization in human sperm from normozoospermic and oligoastenoteratozoospermic patients with or without varicocele. Interesting to note, in samples from patients with varicocele, the expression of steroid receptors is reduced, suggesting a role in the physiopathology of testicular varicocele. Importantly, as a consequence of the low steroid’s receptors expression in varicocele, sperm exhibit a reduced responsiveness to the steroids [38]. Given the important role of steroids on sperm physiology, the missing receptors may cause impaired sperm activity, since in the male genital tract, sperm life is prevalently under the control of the androgens, while in the female genital tract the main hormones are the estrogens and progesterone.

The detrimental effects of varicocele and sperm metabolism: In the human male gamete, given its polarization, the proteins localization may be indicative of their own function. Given the common location of the steroid’s receptors at mitochondrial level, we hypothesized their involvement in energy metabolism, aspect of the gamete biology that was less studied and therefore unclear. Therefore, we treated our samples with physiological activators for each steroid receptor. To highlight the involvement of each receptor in mediating the studied effects, we have made co-treatments with specific inhibitors, ICI for ERs, RU for PRs and Casodex, a specific AR antagonist [32,33,38]. As above mentioned, the sperm, during his lifetime, passes through two physiological stages: A steady state, uncapacitated, during which the gamete economize and / or stores energy, and a state of functional maturation, during which the gamete becomes precisely capacitated with considerable energy expenditure. Generally, it might be briefed that to the uncapacitated gamete is associable an anabolic metabolism, while in the capacitated state to catabolic one. Our previous studies have shown that steroids also induce capacitation, in new studies we observed that, in the gametes from normozoospermic patients, they activate both the carbohydrate and lipidic metabolism. In varicocele, where a reduced or absent response to steroids was observed in each metabolic assay considered, an accumulation of glycogen and triglycerides was obtained. This metabolic condition is like that observed in uncapacitated gametes. Therefore, it may be deduced that the gamete from patient with varicocele have difficulties to switch into capacitation [39,40].

Conclusion and Perspectives

Although, exciting progress has been made over the last years, sperm molecular set up and the regulation of its functions are at the beginning of the knowledge. It will be very hard to fully elucidate the almost unknown molecular sperm composition, however, we think that new studies are needed, since not all the apparently normal spermatozoa are able to fertilize. Furthermore, the functional roles of the great mRNA populations need to be deepened, since it was shown that the presence of some of these mRNAs is essential in the early stages of embryo development.

A recent report showing that an epigenetic alteration mediated by mRNAs to the phenotype, is sperm-derived, supporting the view that the RNA delivered at fertilization can act as an epigenetic modifier of early embryo development.

In conclusion, the paternal contribution to the fertilization process was reassessed since, till now, it was thought that the sperm acts as a cargo only to transport the paternal genome to the oocyte. However, other components of the sperm are essential for normal syngamy in all the investigated mammalian orders (with exception of some rodents). These include an oocyte activating factor (a member of the phospholipase group).

Far from being the quiescent cell we have also seen that spermatozoa are capable to regulate its own functions independently by the systemic regulation and of surprising intranuclear dynamics that includes novel functions for mRNAs. Knowing the messages that the male gamete carry as mRNAs could lead to a redefinition of the paternal and maternal roles both in fertilization and embryo development. Metabolomics and proteome of human spermatozoa were done, however currently the exact role of many proteins in male infertility is unknown. Altogether, we hypothesize that new intriguing discoveries await to be known about the biology of this cell.

Declarations

Data Availability

Not applicable because the study only presents a review of existing data. No new data were generated.

Acknowledgments

We would like to thank Editage (www.editage.com ) for English language editing.

Conflict of Interest

The Authors declare no conflict of interest.

Authors Role

A.S. and V.A. wrote the original draft and edited the manuscript. A.S. and R.V. organized the design and supervised the referenced article. U.L helped to organize, edited and revised the review. P.G. and S. M. contributed to revised the methodology, conceptualization, and the manuscript: S.M. and A.H helped to revised the review. All Authors have read and agreed to the published version of the manuscript.

Funding

The study was funded by MIUR Ex 60%-2024.

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