Composition of Semen: Sperm and Seminal Plasma

The contribution of paternal factors, which, as aforementioned, is at least 30%, comes from the semen. This differs from the case of the female, where the respective contribution of 30–35% does not only involve ovarian dysfunction and advancing maternal age, which have repercussions on oocyte quality and competence, but also problems derived from uterine, pelvic and Fallopian tube alterations (including endometriosis), and other factors such as psychosexual ones (Child Reference Child2013).

The semen is composed of a cellular fraction (sperm) and a liquid one (seminal plasma, also known as seminal fluid) (Figure 2). Regarding the former, three parts can be distinguished in a spermatozoon: head (composed of nucleus and acrosome), neck (or connecting piece), and tail (where mitochondrial, principal and end pieces are distinguished). Remarkably, the spermatozoon is a very peculiar, complex and differentiated cell, as it is the only one that performs its mission (i.e., bring the father haploid genome to the oocyte) in another body (the female’s). It is formed through spermatogenesis, and then completes its maturation during the transit along the epididymis. This transit is featured by the remodelling of the plasma membrane, nuclear compaction, and incorporation of proteins, including cytosine-rich secretory protein 1 (CRISP1) (Cohen et al. Reference Cohen, Maldera, Vasen, Ernesto, Muñoz, Battistone and Cuasnicú2011; Weigel Muñoz et al. Reference Weigel Muñoz, Battistone, Carvajal, Maldera, Curci, Torres, Lombardo, Pignataro, Da Ros and Cuasnicú2018), acrosin binding protein (ACRBP) (Nagdas et al. Reference Nagdas, Hamilton and Samirsubas2010; Yin et al. Reference Yin, Shen, Wan, Liu, Zhou and Zhang2018), and disintegrins/metalloproteinases (ADAM) (Inoue et al. Reference Inoue, Ikawa, Isotani and Okabe2005; Nishimura et al. Reference Nishimura, Myles and Primakoff2007), through extracellular vesicles (James et al. Reference James, Carrell, Aston, Jenkins, Yeste and Salas-Huetos2020; Barrachina et al. Reference Barrachina, Battistone, Castillo, Mallofré, Jodar, Breton and Oliva2022). In addition, motile ability is acquired in the epididymis cauda (Tourzani et al. Reference Tourzani, Battistone, Salicioni, Breton, Visconti and Gervasi2021). As discussed later, epididymosomes also contain non-coding RNAs, and could be involved in passing the paternal epigenetic inheritance to the offspring (Zhang et al. Reference Zhang, Zhang, Shi, Tuorto, Li, Liu, Liebers, Zhang, Qu and Qian2018; Chan et al. Reference Chan, Morgan, Adrian Leu, Shetty, Cisse, Nugent, Morrison, Jašarević, Huang, Kanyuch, Rodgers, Bhanu, Berger, Garcia, Ament, Kane, Neill Epperson and Bale2020).

Figure 2. Semen composition. The semen is composed of cellular (sperm) and liquid (seminal plasma, also known as seminal fluid) fractions. Sperm are produced in the testis through spermatogenesis and then enter the efferent duct and epididymis, where they mature. The seminal plasma (SP) comprises secretions from the testis, epididymis, seminal vesicles, the prostate and bulbourethral glands.

In humans, sperm are deposited in the vagina, and then enter the cervical canal, where they must pass though the cervical mucus. Active and passive (myometrium) contractions let sperm travel along the uterus. In the uterus, sperm trigger NETosis, which is a process through which polymorphonuclear granulocytes, a type of white blood cells, release their DNA into the extracellular environment to generate neutrophil extracellular traps (NETs), which can capture and kill bacteria and sperm (Zambrano et al. Reference Zambrano, Carrau, Gärtner, Seipp, Taubert, Felmer, Sanchez and Hermosilla2016). NETosis appears to have a selective mission, removing the excess of sperm, particularly those that have less motility and potentially less fertility, as studies in animal models indicate (Alghamdi and Foster Reference Alghamdi and Foster2005; Alghamdi et al. Reference Alghamdi, Lovaas, Bird, Lamb, Rendahl, Taube and Foster2009; Zambrano et al. Reference Zambrano, Namuncura, Uribe, Schulz, Pezo, Burgos, Taubert, Hermosilla and Sanchez2021). Those sperm that escape from leukocytes reach the utero-tubal junction (UTJ) (Ishimoto and Gaffney Reference Ishimoto and Gaffney2016). This junction has a selection role, as it presents different folds that allow for the retention of poor-quality sperm. Sperm cells capable of overcoming the UTJ then enter the fallopian tube, where, based on observations performed in animal models and in vitro studies in humans (Pacey et al. Reference Pacey, Hill, Scudamore, Warren, Barratt and Cooke1995; Ziskind et al. Reference Ziskind, Paltieli, Eibschitz, Ohel and Weichselbaum2000; Massa et al. Reference Massa, Prez, Zumoffen, Morente and Ghersevich2019), they appear to attach to the epithelial cells of the isthmus, one of the fallopian tube’s sections, and form a ‘reservoir’ (Sharif et al. Reference Sharif, Hickl, Juarez, Di, Kerns, Sutovsky, Bovin and Miller2022). Only sperm in good shape can bind these epithelial cells; hence, this reservoir has been posited to exert a selective function (Rath et al. Reference Rath, Knorr and Taylor2016), and would drive the transient storage of sperm, maintaining their survival until the oocyte is available (Teijeiro and Marini Reference Teijeiro and Marini2012). Moreover, an in-vitro study conducted in humans reported that binding of sperm to OE-E6/E7, an immortalized human cell line of fallopian tube cells, triggers a transcriptomic response in these epithelial cells. This results in a downregulation of inflammatory molecules (cytokines and chemokines) that ultimately allow for a greater immunological tolerance with regard to sperm (Mousavi et al. Reference Mousavi, Mohammadi, Amjadi, Zandieh, Aghajanpour, Aflatoonian, Sabbaghian, Eslami, Madani and Aflatoonian2021). When, in contrast, sperm incubated with the OE-E6/E7 cell line have high levels of DNA fragmentation (evaluated with the TUNEL assay), they rather activate a toll-like receptor signalling pathway in these cells, upregulating the expression of inflammatory cytokines and chemokines (Mohammadi et al. Reference Mohammadi, Mousavi, Sheibak, Amjadi, Zandieh, Aghajanpour, Aflatoonian, Sabbaghian, Eslami and Aflatoonian2022).

Around ovulation, sperm detach from the epithelial cells of the isthmus and capacitate. Only capacitated sperm are able to trigger the acrosome reaction, which is required for the male gamete to pass through the cumulus cells and the zona pellucida (Molina et al. Reference Molina, Luque, Balestrini, Marín-Briggiler, Romarowski and Buffone2018; Gupta Reference Gupta2021). Some defective sperm cells are unable to undergo capacitation and acrosome reaction, and/or penetrate the oocyte, whereas others – even when they fuse with the oocyte – are not able to activate the oocyte and drive embryo development (Colombero et al. Reference Colombero, Moomjy, Sills, Rosenwaks and Palermo1999). For example, microinjection of parts of the sperm (such as the head or the tail) gives rise to an embryo that is not able to withstand several mitotic divisions (Moomjy et al. Reference Moomjy, Colombero, Veeck, Rosenwaks and Palermo1999).

The following sections scrutinize what we know about sperm components that are potentially involved in the interaction with the oocyte (i.e., gamete fusion), and what happens thereafter (oocyte activation and onset of embryogenesis). This includes: sperm proteome, centrioles, chromatin condensation and DNA integrity, epigenome and metabolome. The seminal plasma is approached in a separate section.

The Sperm Proteome

The sperm proteome differs between fertile and infertile men (Légaré et al. Reference Légaré, Droit, Fournier, Bourassa, Force, Cloutier, Tremblay and Sullivan2014). Castillo et al. (Reference Castillo, Jodar and Oliva2018) conducted a meta-analysis, identifying 103 sperm proteins that could be involved in fertilization and 93 that could play a role during early embryo development. This section is split two different subsections, referring to sperm proteins that could play a role (i) during, and (ii) after fertilization.

Sperm Proteins Involved after Fertilization

Whether the male proteome can contribute to early embryo development has been a subject of research, despite the technical difficulties to address the matter. From the 93 sperm proteins potentially involved in post-fertilization events, 11 have been suggested to have a mission during the first embryo divisions (up to eight-cell stage), 29 in the formation of morulae, and 19 in blastocyst development.

A sperm-borne protein that has been largely confirmed to be relevant for what occurs after gamete fusion is phospoholipase C Zeta (PLCζ), a sperm-specific phospholipase C located in the equatorial and post-acrosomal regions (Figure 3) (Yeste et al. Reference Yeste, Delgado-Bermúdez, Jones and Coward2023). Mounting evidence supports that this protein is involved in oocyte activation, which is the process that releases the oocyte from its metaphase-II arrest, then allows for the onset of embryogenesis, and is featured by Ca²⁺ oscillations in the oocyte cytoplasm (Saunders et al. Reference Saunders, Larman, Parrington, Cox, Royse, Blayney, Swann and Lai2002; Horner and Wolfner Reference Horner and Wolfner2008; Yeste et al. Reference Yeste, Jones, Amdani, Patel and Coward2016, Reference Yeste, Delgado-Bermúdez, Jones and Coward2023). Sperm devoid of PLCζ are unable to trigger Ca²⁺ oscillations, which leads to oocyte activation deficiency and lower fertilization rates (Heytens et al. Reference Heytens, Parrington, Coward, Young, Lambrecht, Yoon, Fissore, Hamer, Deane and Ruas2009; Heindryckx et al. Reference Heindryckx, Nikiforaki, Caluwaerts, vanden Meerschaut, Deroo and de Sutter2013; Kashir et al. Reference Kashir, Jones, Lee, Rietdorf, Nikiforaki, Durrans, Ruas, Tee, Heindryckx and Galione2011, Reference Kashir, Jones, Mounce, Ramadan, Lemmon, Heindryckx, De, Parrington, Turner and Child2013; Yelumalai et al. Reference Yelumalai, Yeste, Jones, Amdani, Kashir, Mounce, da Silva, Barratt, McVeigh and Coward2015). Mutations in the coding sequence of this gene result in an impaired activity of this protein and total fertilization failure (Escoffier et al. Reference Escoffier, Lee, Yassine, Zouari, Martinez, Karaouzène, Coutton, Kherraf, Halouani and Triki2016). A study conducted by Hachem et al. (Reference Hachem, Godwin, Ruas, Lee, Buitrago, Ardestani, Bassett, Fox, Navarrete and De2017) used a Plcz1 knock-out male mouse model and confirmed the crucial role of this protein for oocyte activation, although the sperm from Plcz1 knock-out males were still able to initiate embryogenesis after oocyte fertilization. In addition, total fertilization failure has been found to occur when sperm present the adequate levels of PLCζ. All these data suggest that sperm proteins other than PLCζ could be involved in oocyte activation. For this reason, other sperm proteins, such as post-acrosomal sheath WW domain-binding protein (WBP2NL), have been suggested to play a role during oocyte activation (Wu et al. Reference Wu, Sutovsky, Manandhar, Xu, Katayama, Day, Park, Yi, Yan and Prather2007; Aarabi et al. Reference Aarabi, Qin, Xu, Mewburn and Oko2010, Reference Aarabi, Balakier, Bashar, Moskovtsev, Sutovsky, Librach and Oko2014; Kennedy et al. Reference Kennedy, Krieger, Sutovsky, Xu, Vargovič, Didion, Ellersieck, Hennessy, Verstegen and Oko2014; Kaya et al. Reference Kaya, Dogan, Vargovic, Kutchy, Ross, Topper, Oko, van der Hoorn, Sutovsky and Memili2022). Yet, the exact function of WBP2NL has not been ascertained, as other studies did not observe the same results (Nomikos et al. Reference Nomikos, Sanders, Theodoridou, Kashir, Matthews, Nounesis, Lai and Swann2014, Reference Nomikos, Sanders, Kashir, Sanusi, Buntwal, Love, Ashley, Sanders, Knaggs and Bunkheila2015; Freour et al. Reference Freour, Barragan, Ferrer-Vaquer, Rodríguez and Vassena2017).

Figure 3. Protein phospholipase C zeta (PLCζ). Sperm-specific protein PLCζ is involved in oocyte activation. Upon gamete fusion, it is released into the ooplasm, where it triggers the signalling pathway that alleviates the oocyte from the metaphase-II arrest. Abbreviations: APC, Anaphase-promoting complex/cyclosome; CaM/CaMKII, Calmodulin/Calmodulin-dependent protein kinase II; CSF, Cytostatic factor; CNB1, Cyclin B1; CDK1, Cyclin-dependent kinase 1; DAG, Diacylglycerol; IP3, Inositol 1,4,5-trisphosphate; IP₃R, IP3 receptor; MAPK, Mitogen-activated protein kinase; PIP2, Phosphatidylinositol 4,5-bisphosphate; PKC, Protein kinase C. Reproduced from Yeste et al. (2023) with permission.

Other proteins that could play a role after fertilization are desmocollin 3 (DSC3), which is delivered via sperm to the zygote (Den et al. Reference Den, Cheng, Merched-Sauvage and Koch2006) and has been proposed to regulate cell adhesion in blastomeres before embryo genome activation (EGA) (Castillo et al. Reference Castillo, Jodar and Oliva2018); lactosylceramide 1,3-N-acetyl-beta-D-glucosaminyltransferase (B3GNT5), which would be implied in morula formation (Biellmann et al. Reference Biellmann, Hülsmeier, Zhou, Cinelli and Hennet2008); and choline-phosphate cytidyltransferase A (PCYT1A), which could be implicated in blastocyst development and embryo implantation (Wang et al. Reference Wang and Magdaleno2005).

The Centrioles

Centrioles are crucial organelles in eukaryotic cells, and are involved in the organization of the cytoskeleton, cell division, and formation of flagellum. Mature sperm have two centrioles located in the neck: the proximal (PC) and the distal centriole (DC) (Figure 4) (Khanal et al. Reference Khanal, Leung, Royfman, Fishman, Saltzman, Bloomfield-Gadêlha, Zeev-Ben-Mordehai and Avidor-Reiss2021; Leung et al. Reference Leung, Roelofs, Ravi, Maitan, Henning, Zhang, Bromfield, Howes, Gadella and Bloomfield-Gadêlha2021). In most eutherian mammals except rodents, paternal centrioles are inherited by the fertilized oocyte, which does not have centrioles. For this reason, paternal centrioles organize the cytoskeleton of the zygote, mediate pronuclei migration (Scheffler et al. Reference Scheffler, Uraji, Jentoft, Cavazza, Mönnich, Mogessie and Schuh2021) and drive cell cleavage (Rawe et al. Reference Rawe, Terada, Nakamura, Chillik, Olmedo and Chemes2002; Amargant et al. Reference Amargant, Pujol, Ferrer-Vaquer, Durban, Martínez, Vassena and Vernos2021; Avidor-Reiss et al. Reference Avidor-Reiss and Fishman2019; Reference Avidor-Reiss, Achinger and Uzbekov2022). Sperm from infertile men have been identified to have abnormal centrioles (Turner et al. Reference Turner, Fishman, Asadullah, Ott, Dusza, Shah, Sindhwani, Nadiminty, Molinari and Patrizio2021), and the fact that paternal centrioles in humans have a crucial function after fertilization has been suggested to be one of the reasons for the greater proportion of aneuploid embryos in humans compared with rodents (Cannarella et al. Reference Cannarella, Condorelli, Mongioì, La Vignera and Calogero2020).

Figure 4. Sperm centrioles. Centrioles are crucial organelles in eukaryotic cells, which are involved in the organization of the cytoskeleton, cell division, and flagellum formation. Mature sperm have two centrioles located in the connecting piece: the proximal (PC) and the distal centriole (DC) (A). After gamete fusion (B), the sperm centrosome forms an aster while the oocyte completes meiosis II and the second polar body is extruded (C). Thereafter, centrioles begin to duplicate to produce two daughter centrioles (D), which is followed by the separation of the zygote centrosomes (E). Subsequently, the zygote undergoes mitosis (F), leading to the formation of two blastomeres. Abbreviations: Ax, Axoneme; Ca, Centriole adjunct; DC, Distal centriole; PC, Proximal centriole; PCL, Proximal centriole-like structure; ZdC, Zygotic daughter centriole; PB, Polar body; N, Ploidy. Reproduced from Vallet-Buisan et al. (Reference Vallet-Buisan, Mecca, Jones, Coward and Yeste2023) with permission.

The Relevance of Nuclear Integrity: Chromatin and DNA

In contrast to prokaryotic cells, DNA is bound to proteins in the eukaryotic ones forming a structure called chromatin, which, during interphase, is isolated from the rest of the cytoplasm thanks to the nuclear envelope. While chromatin is formed by DNA and histones (which may bear epigenetic signatures) in somatic cells (Bartosovic et al. Reference Bartosovic, Kabbe and Castelo-Branco2021), mature sperm get most of these histones (85–90%) replaced by protamines (protamine 1 and protamine 2, in the case of humans) (Figure 5) (Brykczynska et al. Reference Brykczynska, Hisano, Erkek, Ramos, Oakeley, Roloff, Beisel, Schübeler, Stadler and Peters2010; Jung et al. Reference Jung, Sauria, Lyu, Cheema, Ausio, Taylor and Corces2017). This occurs during spermiogenesis, when the canonical organization of chromatin with nucleosomes changes to a toroidal one around protamines (Barral et al. Reference Barral, Morozumi, Tanaka, Montellier, Govin, Charbonnier, Couté, Puthier and Buchou2017). The different toroids, each containing about 200 kb of DNA, are linked with toroid-linker regions, which contain histones and mediate the attachment to the proteinaceous nuclear matrix (Narwade et al. Reference Narwade, Patel, Alam, Chattopadhyay, Mittal and Kulkarni2019). Unlike histones, protamines contain arginine to allow for stronger binding to DNA in order to form a toroid-like ridged structure. As sperm do not have all the machinery required for repairing DNA damage, the greater degree of condensation provided by protamines has been suggested as a strategy to maintain DNA integrity better (Ni et al. Reference Ni, Spiess, Schuppe and Steger2016). Remarkably, the 10–15% of histones retained in the sperm chromatin may bear epigenetic marks, such as H3K23me, and are organized in nucleosomes (Champroux et al. Reference Champroux, Cocquet, Henry-Berger, Drevet and Kocer2018). The potential contribution of these epigenetic signatures brought into the zygote by sperm is discussed later (see the section about the sperm epigenome).

Figure 5. (a) Chromatin remodelling during spermiogenesis. Some histones are replaced with testis-specific histone variants. Histones then undergo post-translational modifications. Following this, histones are replaced by transition nuclear proteins, and these by protamines, which ultimately increases chromatin packaging. (b) Organization of chromatin in sperm. In sperm, chromatin is organized into three components: the most abundant, which consists of DNA bound to protamines; histone-bound DNA; and nuclear matrix attachment regions (MAR). Protamine-bound DNA is coiled into toroids, and histone-bound DNA is present in retained solenoids and toroid linkers. Abbreviations: MAR, Matrix attachment region; PTM, post-translational modification; P1, protamine 1; P2, protamine 2; TNP, transition nuclear protein. Reproduced from Balder et al. (Reference Balder, Jones, Coward and Yeste2024) with permission.

The Sperm Transcriptome

Although mammalian sperm are widely regarded as transcriptionally silent cells and they have about 600 times less mRNA than somatic cells (Zhao et al. Reference Zhao, Li, Yao, Wang, Zhou, Wang, Liu, Wang, Wang and Qiao2006; Ren et al. Reference Ren, Chen, Wang and Wang2017), their nuclei exhibit residual DNA and RNA polymerase activity (Bianchi et al. Reference Bianchi, Stermer, Boekelheide, Sigman, Hall, Reyes, Dere and Hwang2018). Whether sperm transcripts could play a function is, however, not clear. Many have suggested that sperm mRNAs could be delivered to the oocyte, where they could be involved in the onset of embryogenesis, perhaps before embryo genome is activated (Ko et al. Reference Ko, Kitchen, Wang, Threat, Wang, Hasegawa, Sun, Grahovac, Kargul and Lim2000; Hayashi et al. Reference Hayashi, Yang, Christenson, Yanagimachi and Hecht2003; Ostermeier et al. Reference Ostermeier, Miller, Huntriss, Diamond and Krawetz2004; Jodar et al. Reference Jodar, Sendler, Moskovtsev, Librach, Goodrich, Swanson, Hauser, Diamond and Krawetz2015; Ntostis et al. Reference Ntostis, Carter, Iles, Huntriss, Tzetis and Miller2017). Different sperm-borne transcripts, such as PSG1 and HLA-E, have been hypothesized to be involved in embryo implantation (Avendaño et al. Reference Avendaño, Franchi and Oehninger2008), perhaps because they play an immunotolerance function, with the sperm levels of PSG1 and HLA-E mRNA being higher in fertile than in infertile men (Avendaño et al. Reference Avendaño, Franchi, Jones and Oehninger2009). Other transcripts, such as SSFA2 and SESN1, have been suggested to be involved in the development of pig embryos until the 4-cell stage, when EGA occurs (Guo et al. Reference Guo, Chao, Xiao, Wang, Meng, Li, Han, Ouyang, Hou and Sun2017). Embryo development is also related to the levels of protamine-1 (PRM1), protamine-2 (PRM2), POU domain class 5 transcription factor 1 (POU5F1, also known as OCT4), glutathione peroxidase 4 (GPX4), and heat shock protein 90 (HSP90AA1) transcripts in sperm (Cho et al. Reference Cho, Jung-Ha, Willis, Goulding, Stein, Xu, Schultz, Hecht and Eddy2003; Meseguer et al. Reference Meseguer, de los Santos, Simón, Pellicer, Remohí and Garrido2006; Hwang et al. Reference Hwang, Mulligan, Kim, Yang, Lee, Hwang, Mulligan, Kim, Yang and Lee2013; Rogenhofer et al. Reference Rogenhofer, Ott, Pilatz, Wolf, Thaler, Windischbauer, Schagdarsurengin, Steger and von Schönfeldt2017; Sadakierska-Chudy et al. Reference Sadakierska-Chudy, Patrylak, Janeczko and Chudy2020). As most of these studies assume that sperm transcripts play a role during embryogenesis because they are present in the embryo, further research to ascertain their specific function is needed. It also remains unaddressed when and how they are degraded, as in the mouse this happens as early as the zygote stage (Hayashi et al. Reference Hayashi, Yang, Christenson, Yanagimachi and Hecht2003).

The Sperm Epigenome

Epigenetics involve a series of regulatory mechanisms that modify gene expression without altering the DNA. In sperm, the epigenome includes DNA methylation, histone modifications and non-coding RNAs (Figure 6); all these elements can also contribute to embryo development (Smith et al. Reference Smith, Chan, Humm, Karnik, Mekhoubad, Regev, Eggan and Meissner2014).

Figure 6. Epigenetic signatures in sperm. The contributors to the sperm epigenome include modifications to DNA and histones, RNAs and protamines. Abbreviations: lncRNA, long noncoding RNA; miRNA, microRNA; piRNA, piwi-interacting RNA; PTMs, post-translational modifications; siRNA, small interfering RNA. Reproduced from Balder et al. (Reference Balder, Jones, Coward and Yeste2024) with permission.

The Sperm Metabolome

Metabolomics has been emerging over the last decade and has been proved to have many applications. Most of the studies focused on semen investigated the metabolites of seminal plasma. One of these studies revealed that men suffering from oligoasthenospermia had significantly lower values of free L-carnitine, polyunsaturated fatty acids, glutamate, aspartate, methionine, tryptophan, proline, and alanine; and four biogenic amines (spermine, spermidine, serotonin, and alpha-aminoadipate). This deficiency in L-carnitine could be related to a decreased activity of β-oxidation of fatty acids, and oxidative phosphorylation, whereas reduced levels of amino acids and biogenic amines could be related to dysregulated signalling pathways (Boguenet et al. Reference Boguenet, Bocca, Bouet, Serri, Chupin, Tessier, Blanchet, El Hachem, Chao de la Barca, Reynier and May-Panloup2020).

Other research, which also targeted seminal plasma, compared fertile and infertile patients and identified up to 40 molecules (metabolomics/lipidomics approach) differing between fertile and infertile men. Acylcarnitines, phosphatidylserine (PS) (40:2) and lactate were lower, and PE (18:1; 18:1), phosphatidic acid (PA) (O-19:2; 18:1), lysophosphatidylethanolamine (LPE) (O-16:1) and phosphatidylcholine (PC) (O-16:2; 18:1)-CH3 were higher in infertile patients (Correnti et al. Reference Correnti, Preianò, Fregola, Gamboni, Stephenson, Savino, D’Alessandro and Terracciano2023). In a systematic review, Llavanera et al. (Reference Llavanera, Delgado-Bermúdez, Ribas-Maynou, Salas-Huetos and Yeste2022) compiled a list of metabolites whose levels in seminal plasma were significantly different between fertile and infertile men, including lactate, alanine, choline, citrate, glycero-phosphocholine, glutamine, tyrosine, histidine, phenylalanine, and uridine. Another study found that smoking alters the metabolism of sperm, which could potentially affect fertilizing ability, as it decreases the uptake of fatty acids by sperm mitochondria, which in turn decreases energy supply (Engel et al. Reference Engel, Baumann, Blaurock, Rolle-Kampczyk, Schiller, von Bergen and Grunewald2021).

On the other hand, and given the events occurring during gamete fusion and thereafter, it seems quite unrealistic that sperm metabolites have a direct influence on fertilization and early embryo development. In spite of this, Guo et al. (Reference Guo, Li, Hao, Yang, Yang, Chang, Kong, Liu, Jiao and Teng2023) compared sperm from young and aged men, and identified 129 differentially expressed metabolites; from these, four, such as pipamperone, 2,2-bis(hydroxymethyl)-2,2’,2’’-nitrilotriethanol, Arg-Pro and triethyl phosphate, were more abundant in aged men. These findings are supported by investigations performed in animal models. In cattle, a previous study found that from 3704 metabolites identified in sperm, bulls with different fertility differed in the levels of 33 metabolites. These metabolites were related to taurine and hypotaurine, whose levels were reduced in bulls with low fertility, and also with different routes, such as glycolysis, β-oxidation of fatty acids, and synthesis of pyrimidines (Talluri et al. Reference Talluri, Kumaresan, Sinha, Paul, Ebenezer Samuel King and Datta2022).

Also in cattle, a study using liquid chromatography–mass spectrometry (LC-MS) identified up to 3704 metabolites in sperm, and identified five metabolites (hypotaurine, selenocysteine, l-malic acid, d-cysteine, and chondroitin 4-sulfate) that differed between bulls of high and low fertility (Saraf et al. Reference Saraf, Kumaresan, Dasgupta, Karthikkeyan, Prasad, Modi and Ramesha2020). In pigs, sperm with higher quality and fertilizing ability, and giving rise to more embryos at day 6 having greater levels of glycolysis-derived metabolites than those with poor quality, were found to have greater amounts of metabolites related to oxidative phosphorylation. This suggests the basal metabolism in mammalian sperm could be relevant for the ability of sperm to fertilize the oocyte and give rise to a viable embryo (Mateo-Otero et al. Reference Mateo-Otero, Madrid-Gambin, Llavanera, Gomez-Gomez, Haro, Pozo and Yeste2023). Finally, another study also conducted in pigs found that alteration of sperm mitochondrial activity, which impacts oxidative phosphorylation and metabolism, could increase ROS levels, which could in turn affect sperm DNA integrity and ultimately embryo development (Mateo-Otero et al. Reference Mateo-Otero, Llavanera, Torres-Garrido and Yeste2024).

All this compiled evidence, therefore, indicates that sperm metabolic fate influences fertilizing ability and could be even relevant while giving rise to a viable embryo, as the yet few studies in humans and animal models suggest.

Does the Seminal Plasma have Any Role?

Seminal plasma is the liquid part of semen, and is produced by epididymis and male accessory glands (prostate, seminal vesicles) (Figure 7). While this fluid has largely been regarded as a mere vehicle for sperm upon ejaculation, growing evidence supports that it interacts with the female reproductive tract and can be involved in the modulation of the uterine environment, embryo development, and foetal growth (Bromfield et al. Reference Bromfield, Schjenken, Chin, Care, Jasper and Robertson2014; Watkins et al. Reference Watkins, Dias, Tsuro, Allen, Emes, Moreton, Wilson, Ingram and Sinclair2018). In mice, seminal plasma modulates the expression of tumour necrosis factor (TNF), interleukin-1β (IL-1β), interferon-γ (IFN-γ), microphage inflammatory protein 1-α (MIP-1α) and colony stimulating factor 3 (CSF3), and the expression of regulatory T-cell genes (Watkins et al. Reference Watkins, Dias, Tsuro, Allen, Emes, Moreton, Wilson, Ingram and Sinclair2018) in the endometrium, which supports its immunomodulation role. This would increase the maternal tolerance to paternal and foetal antigens, and facilitate embryo implantation in mice (Robertson et al. Reference Robertson, Mau, Tremellen and Seamark1996, Reference Robertson, Sjöblom, Jasper, Norman and Seamark2001; Bromfield et al. Reference Bromfield, Schjenken, Chin, Care, Jasper and Robertson2014).

Figure 7. Origin, components, and functions of seminal plasma (SP). Seminal plasma represents more than 95% of the semen volume, whereas testicular secretions containing sperm account for 2–5%. The seminal plasma is composed of a complex set of heterogeneous molecules, such as proteins (enzymes, cytokines, TEX101, ACRV1, TGFββ, prostate-specific antigen (PSA), prostatic-specific acid phosphatase (PSAP), etc.), lipids, sugars (fructose), cell-free nucleic acid (DNA, microRNA, and LncRNA), ions (Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, etc.), and small-molecule metabolites. Not only does SP modulate sperm function, but some of its components, such as cytokines, also recognize receptors on epithelial cells lining the cervix and uterus and induce the synthesis of pro-inflammatory cytokines and chemokines that recruit and activate inflammatory leukocytes. The SP also modulates the release of cytokines and growth factors, which appear to regulate embryo development in the oviduct and uterus before implantation. Reproduced from Wang et al. (Reference Wang, Cheng, Xia, Liu, Guo, Yang, Guo and Xu2020), which was published under the terms and conditions of a Creative Commons Attribution license (CC BY 4.0).

Since humans do not ejaculate in the uterus, as mice do, but in the vagina, the role that seminal plasma could play in this species is less clear, as it remains unknown whether it reaches the uterus. Studies in other animal models that, like cattle, also ejaculate in the vagina indicate that seminal plasma factors could adhere onto the sperm surface, thereby influencing the uterine environment (Recuero et al. Reference Recuero, Sánchez, Mateo-Otero, Bagés-Arnal, McDonald, Behura, Spencer, Kenny, Yeste and Lonergan2020), as in this species they also induce the production of cytokines and chemokines in the female genital tract (Schjenken et al. Reference Schjenken, Glynn, Sharkey and Robertson2015; Nongbua et al. Reference Nongbua, Guo, Ntallaris, Rubér, Rodriguez-Martinez, Humblot and Morrell2020). These findings would match those in humans, where seminal plasma has been proposed to prime the maternal endometrium for implantation (Ibrahim et al. Reference Ibrahim, Rizo, Fontes, Lamb and Bromfield2019; George et al. Reference George, Jang, Nyegaard, Neidleman, Spitzer, Xie, Chen, Herzig and Laustsen2020; Ajdary et al. Reference Ajdary, Ashrafi, Aflatoonian and Mehdizadeh2021), as it increases pregnancy rate after embryo transfer (Wolff et al. Reference Freour, Barragan, Ferrer-Vaquer, Rodríguez and Vassena2009; Chicea et al. Reference Chicea, Ispasoiu and Focsa2013; Friedler et al. Reference Friedler, Ben-Ami, Gidoni, Strassburger, Kasterstein, Maslansky, Komarovsy, Bern, Ron-El and Raziel2013; Crawford et al. Reference Crawford, Ray, Gudi, Shah and Homburg2015).

Seminal plasma contains extracellular vesicles, mainly epididymosomes and prostasomes. These vesicles are membrane-bound particles released by cells (Yáñez-Mó et al. Reference Yáñez-Mó, Siljander, Andreu, Zavec, Borràs, Buzas, Buzas, Casal, Cappello and Carvalho2015). They have been found to be synthesized by the epididymis and prostate. In the case of the epididymis, these extracellular vesicles, called epididymosomes, fuse with epididymal sperm (Păunescu et al. Reference Păunescu, Shum, Huynh, Lechner, Goetze, Brown and Breton2014). Thanks to this fusion, epididymal cells can transfer proteins and miRNA to sperm (Twenter et al. Reference Twenter, Klohonatz, Davis, Bass, Coleman, Bouma and Bruemmer2020). Proteins identified in these extracellular vesicles include enzymes, and others having a function for sperm motility (Barrachina et al. Reference Barrachina, Battistone, Castillo, Mallofré, Jodar, Breton and Oliva2022), and even galactin-3, which is a lectin transferred from the seminal plasma to the sperm surface, that is involved in the binding of capacitated sperm to the zona pellucida (Mei et al. Reference Mei, Chen, Lee, Zhao, Wang, Lam, Ho, Yeung, Fang and Chiu2019).

Prostasomes, which are produced by prostate epithelial cells (Brody et al. Reference Brody, Ronquist and Gottfries1983), also transfer proteins to sperm (Ronquist et al. Reference Ronquist, Larsson, Ronquist, Isaksson, Hreinsson, Carlsson and Stavreus-Evers2011), and have been suggested to modify the composition of sperm plasmalemma (Dubois et al. Reference Dubois, Ronquist, Ek, Ronquist and Larsson2015). Prostasomes cargo also seems to regulate sperm motility (Andrews et al. Reference Andrews, Galileo and Martin-DeLeon2015; García-Rodríguez et al. Reference García-Rodríguez, la Casa, Peinado, Gosálvez and Roy2018), capacitation (Pons-Rejraji et al. Reference Pons-Rejraji, Artonne, Sion, Brugnon, Canis, Janny and Grizard2011; Aalberts et al. Reference Aalberts, Sostaric, Wubbolts, Wauben, Gadella, Stout and Stoorvogel2013), and even protect sperm from the female immune system (Milutinović et al. Reference Milutinović, Goč, Mitić, Kosanović and Janković2019; Paktinat et al. Reference Paktinat, Hashemi, Ghaffari Novin, Mohammadi-Yeganeh, Salehpour, Karamian and Nazarian2019). In humans, seminal plasma vesicles have been suggested to fuse with endometrial stromal cells, induce decidualization, and increase the secretion of prolactin (Rodriguez-Caro et al. Reference Rodriguez-Caro, Dragovic, Shen, Dombi, Mounce, Field, Meadows, Turner, Lunn and Child2019). All these data indicate that these seminal plasma vesicles play a role with regard to both sperm function and the female reproductive tract, and should be further investigated in the future.