Sperm Interaction with the Female Reproductive Tract
Author’s address (for correspondence): X Druart, UMR 6175 INRA, CNRS-Université de Tours-Haras Nationaux, Station de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. E-mail: email@example.com
Sperm transit in the female tract is a critical event for the success of fertilization. From their deposition in the vagina to final migration in the oviduct, sperm pass through the different compartments of the genital tract in which they encounter different environments. The cervix and the uterotubal junction (UTJ) are two barriers with different relative importance according to the species. The protein composition, the degree of glycosylation and the hydration of the cervical mucus change during the oestrous cycle. Several sperm surface proteins are associated with their migration through the cervical mucus and the UTJ. Data regarding the interaction of sperm with secretions of the epithelial tissue lining the different compartments of the female genital tract during the sperm transit are reviewed, with a particular emphasis on the migration of sperm through the cervix.
Soon after their deposition near the cervix during ejaculation, sperm migrate through the cervix, the uterus and the uterotubal junction (UTJ) to establish a functional population in the caudal isthmus of the oviduct, the site of the sperm reservoir (Hunter et al. 1980, 1982; Nishimura et al. 2004). Several key anatomical regions represent physical barriers to the progression of sperm through the tract. In the majority of the mammalian species, the cervix and the UTJ play a role in the selection of sperm. The sheep cervix contains a series of 5–7 funnel-shaped rings that are not concentrically aligned (Halbert et al. 1990). The morphology of the opening of the cervix is highly variable between females and can be classified as slit, papilla, duckbill, flap or rose (Kershaw et al. 2005). The morphology of the cervix is also variable between sheep breeds with different levels of fertility regarding the length of the cervix and the number of rings (Kaabi et al. 2006). However, to date, little information is available concerning the relation between cervical morphology, secretion activity and sperm migration.
Secretions of the Cervix
The cervical mucus plays a central role in cervical function by controlling sperm migration. Cervical mucus is a gel made up of large glycosylated glycoproteins named mucins with highly glycosylated domains separated by hydrophobic peptide chains (Carlstedt and Sheehan 1989; Gipson 2001). Mucins are secreted by the cervical epithelium and cover the surface of the cervical folds. In ovine, caprine and bovine species, different types of mucin are regionally secreted. Sulfomucins are mainly found on primary folds, whereas sialomucins are found in the secondary indentations (Heydon and Adams 1979; Pluta et al. 2011). Mucus consists predominantly of sialomucins and to a lesser extent, sulfomucins (Heydon and Adams 1979). In ovariectomized ewes, the production of mucus is limited and consists primarily of sulfomucins. The production of mucins is under endocrine control as supplementation with oestradiol benzoate increases the production of mucus, increases the water content and restores the production of sialomucins (Adams and Tang 1979, 1986). The secretions of the cervix are subject to morphological and biochemical changes during the oestrous cycle, as the amount of mucus and its degree of hydration increase considerably during oestrus (Katz et al. 1997).
In humans, the endocervical epithelium expresses mRNA of three of the large gel-forming mucins (MUC5AC, MUC5B and MUC6), with mRNA of MUC5B predominating (Gipson et al. 1997, 1999). The amount of the MUC5B protein in mucus was assessed during the oestrous cycle and was shown to peak around the time of ovulation (Gipson et al. 2001). This increased secretion of MUC5B is associated with modifications of its O-glycosylation during the cycle. MUC5B carries two types of oligosaccharides, the T antigen and N-acetyllactosamine oligosaccharides (Argueso et al. 2002). The amounts of T antigen and N-acetyllactosamine oligosaccharides on MUC5B increased during the first half of the cycle, peaked at midcycle and dropped at the end of the cycle. A proteomic analysis of cervical mucosal secretions before and during ovulation lead to the identification of nearly 200 proteins, including the three gel-forming mucins previously identified, MUC5B, MUC5AC and MUC6 and two transmembrane mucins, MUC16 and MUC1 (Andersch-Björkman et al. 2007). The type and degree of glycosylation of the mucins was also compared before and during ovulation. Cervical mucus at ovulation was characterized by a relative increase in neutral fucosylated oligosaccharides. In the bovine, patterns of glycosylation of mucins in the mucus also changed during the oestrous cycle and were associated with changes in glycosidase activity (Pluta et al. 2011). The role of O-glycans on mucins might be to hold water within the endocervical canal during ovulation to facilitate sperm migration (Argueso et al. 2002) as this is linked to the proportion of water in the mucus (Lee et al. 2002).
In addition to changes in the proportion of mucins or glycosylation, a change in mucus structure during ovulation was proposed (Brunelli et al. 2007). Whereas mucus is arranged in compact fibre-like structures in the pre-ovulatory phase, it was shown, using atomic force microscopy, that ovulatory mucus was composed of floating globules of mucin aggregates (Brunelli et al. 2007). This structure could account for the increased mucus permeability to sperm during ovulation. The switch from globular-ovulatory to fibrous-pre-ovulatory mucus might be driven by pH changes during ovulatory cycles.
So far, comprehensive proteomic studies of cervico-vaginal fluid have been performed only in the human in various physiological and pathological conditions. Protein composition was studied in healthy cyclic women (Andersch-Björkman et al. 2007; Shaw et al. 2007), mid-term pregnant women (Dasari et al. 2007; Gravett et al. 2007) and human papilloma virus infected women (Zegels et al. 2009), and different patterns were observed associated with physiological state. Furthermore, these studies revealed, outside of mucins, a growing complexity of protein composition with more than 800 proteins identified. Mucus protein composition in domestic species is largely unknown and requires further investigation to begin to elucidate biochemical interaction between sperm and cervical mucus.
Sperm Migration Through the Cervix
A very limited amount of data are available concerning a putative role on the migration through the cervix of a biochemical interaction of the sperm with the cervical mucus. To date, the only example is beta-defensin 126 (DEFB126), a glycoprotein shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis (Yudin et al. 2005b). This interaction is considered a major glycocalyx barrier to the external environment and is retained until the completion of capacitation (Yudin et al. 2005a). DEFB126 facilitates the penetration of cervical mucus by sperm (Tollner et al. 2008). It was proposed that DEFB126 imparts a highly negative surface charge to macaque sperm that could be essential for cervical mucus penetration (Tollner et al. 2008). Sperm from men carrying a homozygous variant of the DEFB126 gene exhibited a reduced rate of penetration of synthetic cervical mucus (Tollner et al. 2011). DEFB126 could therefore be involved in the penetration of cervical mucus by sperm in primates.
Technological processing of semen, such as freezing and thawing or liquid storage, alters the ability of sperm to migrate through the female tract, especially through the cervix. In sheep, where fertility levels with frozen semen after laparoscopic artificial insemination (AI) can reach 50–70%, fertility after cervical AI can fall to 10–30% (Maxwell and Salamon 1993; Maxwell and Stojanov 1996; Salamon and Maxwell 2000). Using in vivo imaging, we were able to detect alterations of ram sperm transit in the ewe genital tract after liquid storage (Druart et al. 2009). When fresh and liquid stored ram sperm were cervically inseminated, the proportion of sperm crossing the cervix and reaching the uterus 4 h after deposition was dramatically reduced for stored semen. When sperm were deposited in the lower part of the uterine horn, the proportion of sperm reaching the oviduct and the mobility of the sperm in situ were also reduced for liquid stored semen. However, fertility can be increased when cervical inseminations of liquid stored semen are performed 5 h earlier in relation to the period of ovulation when compared to inseminating with fresh semen (Fernandez-Abella et al. 2003). This reduction in fertility after sperm storage is perhaps because the cells are not able to perform a complete migration through the genital tract and also because it requires more time for them to reach the oviduct near the ovulation period in synchronized cycles.
Differences in fertility between sheep breeds have been observed after cervical AI using frozen-thawed semen. In contrast to the majority of sheep breeds, the fertility of the Norwegian cross-breed after cervical AI is remarkably high, either with 24 h liquid stored semen (Paulenz et al. 2010) or with frozen semen (Paulenz et al. 2004, 2007; Nordstoga et al. 2009). While fertility after laparoscopic AI was similar between Belclare and Suffolk breeds, fertility after cervical AI was higher in the Belclare than the Suffolk, suggesting that sperm transit through the cervix was more efficient in the Belclare breed (Fair et al. 2005; O’Hara et al. 2010). Indeed, sperm penetration through cervical mucus was assessed in these two breeds and proved to be higher in the Belclare breed and this could be linked to the rheological properties of mucus (Richardson et al. 2011). Sheep breeds with marked differences in the ability of sperm to penetrate the cervix could be useful models to investigate the mechanisms involved in sperm transit through the cervix.
From the UTJ to the Oviduct
The UTJ is a functional barrier between the uterus and the oviduct, selecting sperm with normal mobility. In addition to mobility, sperm membrane properties are also involved in the crossing of the UTJ. Null mouse mutants for at least seven different genes are infertile because their sperm cannot pass the UTJ or bind to the zona pellucida, despite normal sperm mobility and morphology. These genes include the following: Calmegin (Ikawa et al. 2001), Calreticulin3 or calsperin (Ikawa et al. 2011), Angiotensin-converting enzyme (Hagaman et al. 1998), Adam1a or fertilin α (Nishimura et al. 2004), Adam2 or fertilin β (Cho et al. 1998), Adam3 or cyritestin (Yamaguchi et al. 2009), PGAP1 (Post-GPI Attachment to proteins 1) (Ueda et al. 2007) and PDILT (Tokuhiro et al. 2012). Calmegin and calsperin are testicular isoforms of calnexin and calreticulin, chaperone proteins that assist in the proper folding of proteins and their placement on the cell membrane (Bedard et al. 2005). Calmegin is required for the assembly of the heterodimeric complex Adam1a/Adam2 (Ikawa et al. 2001) and both calmegin and calsperin are required for the presence of Adam3 on the sperm surface (Yamaguchi et al. 2006; Ikawa et al. 2011). The six mutants for Clgn, Ace, Adam1a, Adam2, PDILT and Adam3 share a common feature, the absence or the dislocation of Adam3 in the detergent-rich membrane domain (Yamaguchi et al. 2009; Tokuhiro et al. 2012). Therefore, Adam3 is suggested to be an important factor for sperm migration in the mouse (Ikawa et al. 2010). The precise mechanism by which Adam3 facilitates the passage of sperm through the UTJ is unknown. Interestingly, all these mutants are unable to migrate to the oviduct and bind to the zona pellucida, suggesting that similar mechanisms might be involved in sperm transit and zona binding. The involvement of Adam3 or another sperm membrane protein in the crossing of the UTJ is unknown in other species.
Sperm Interaction with Oviductal Receptors
Upon their transit through the female genital tract, sperm bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Several studies have focused on the identification of sperm oviductal receptors and there is a growing list of candidate proteins.
Ram sperm bind to oviductal epithelial cells (OEC) and remain viable for several hours before fertilization. When ram sperm were cocultured in vitro on OEC monolayers, sperm binding and survival was maintained at least for 48 h (Lloyd et al. 2008). In pigs, coculture of sperm with OEC also improved their survival (Yeste et al. 2009). The conditioned medium from OEC culture was able to improve sperm survival but maximal effect was only observed when sperm where in direct contact with the OEC (Yeste et al. 2009).
The survival of sperm cells was improved when incubated with apical membrane extracts isolated from oviduct epithelial cells, in the boar (Fazeli et al. 2003), bull (Boilard et al. 2004) and ram (Lloyd et al. 2009). HSPA8 was identified among the protein components of the ewe oviduct extracts (Lloyd et al. 2008) and recombinant HSPA8 was able to improve sperm survival in the bull, ram and boar (Elliott et al. 2009; Lloyd et al. 2009).
Two chaperone proteins, Hsp60 and GRP78, were shown to be present on the apical membrane of bovine and human OEC, and these are able to bind to the sperm membrane (Boilard et al. 2004; Lachance et al. 2007). Hsp60 and GRP78 improved sperm survival in the bull (Lachance et al. 2007). In addition, GRP78 was found in human oviductal fluid during the periovulatory period (Marin-Briggiler et al. 2010) and may be involved in sperm capacitation and zona pellucida binding (Lachance et al. 2007; Marin-Briggiler et al. 2010).
Upon ejaculation, seminal vesicle proteins associate with the sperm membrane and this might affect sperm interaction with the female genital tract. Binding of ejaculated bull sperm to the oviductal epithelium is promoted by seminal vesicle proteins (PDC109, BSPA3 and BSP30K) that coat the sperm head by associating with plasma membrane phospholipids (Hung and Suarez 2010; Suarez 2008; Gwathmey et al. 2006). Putative oviductal receptors for these seminal vesicle proteins are members of the annexin protein family (Ignotz et al. 2007). Three members of the annexin family have also been found in the pig oviduct (Teijeiro et al. 2009), suggesting this mechanism might be shared between species.
During epididymal maturation and ejaculation, sperm acquire their fertilizing ability through interaction with epididymal secretions and seminal plasma components. The acquisition of fertility includes the ability to migrate through the female genital tract and reach the oocyte in the oviduct. During this transit in the female tract, sperm encounter different environments with various mechanical and biochemical properties. To complete the journey, each sperm has to swim in a highly viscous medium in the cervix, avoid immunological detection in the uterus, cross the UTJ, bind to cells in the oviductal reservoir and be released in time to reach the oocyte. Several proteins from the sperm membrane have been involved in the achievement of this transit. The migration of sperm through the cervix is improved, in primates, by the beta-defensin 126, a protein of epididymal origin, and the crossing of the UTJ requires Adam3, a testicular protein processed during epididymal maturation. Later, the interaction of sperm with the oviduct is facilitated, in the bovine, by the binding of seminal plasma proteins such as PDC109 to the sperm surface. If sperm undergo modifications under the influence of epithelial secretions (Killian 2011), a dialogue between the sperm and the female tract is established, as shown by studies indicating modified secretions of the oviduct in the presence of sperm (Georgiou et al. 2005, 2007). The identification of the participants in this dialogue in vivo will require further research, and this will be important to properly elucidate the mechanisms by which sperm transit and survive in the female genital tract.
Conflicts of interest
None of the authors have any conflicts of interest to declare.