Background
The burgeoning population is a serious concern and contraception is considered the most accepted way of controlling it [
1]. Female contraceptive methods include intrauterine devices (IUDs), barriers, sterilization, hormone-based treatment (oral/injectable) and insert contraceptives. Out of these, the most well-known are hormone-based contraceptives. Despite the fact that these have great contraceptive efficacy and reversibility, they have many side effects viz. spotting and irregular bleeding, weight increase, queasiness, and mood alterations; are irritating and exceptionally troubling sometimes, which lead to their discontinuation [
2]. To some degree, distinctive yet similar issues lead to considerably less use of IUDs as they may lead to infection, heavy menstrual bleeding, dysmenorrhea, and pain during insertion [
3]. Likewise, barrier methods of contraception are though effective but also suffer from certain side effects such as difficulty in insertion, allergic reactions to latex or polyurethane, vaginal irritation, cervical erosion, urinary tract infections and rarely toxic shock syndrome, if the device is left set up for a really long time [
4]. Sterilization is an excellent choice for women who truly want to terminate childbearing, but being an irreversible method, there is evidence of some regret, particularly among women sterilized at younger ages [
5]. Every contraceptive method in use today has shortcomings, and collectively they leave major voids in the ability of people to control fertility safely, effectively, and in culturally acceptable ways throughout their reproductive life. Therefore, there is need to provide woman with safe and effective alternatives.
Commercially available vaginal contraceptive formulations contain non-ionic surfactants such as nonoxynol-9 (N-9), as an active ingredient that causes irreversible immobilization of human spermatozoa but affects vaginal microflora and the epithelial cells when used frequently [
6]. Non-invasive contraceptive agents with spermicidal activity are acrylophenones, vanadocenes, gel microemulsions (GM-4, GM-44), sylidines, thymols, and isoxazoles/isoxazolines [
7]. Amongst the natural products, saponins isolated from the fruit pericarp of
Sapindus mukorossi [
8]
, magainin-A from the skin of the African clawed frog
Xenopus laevis [
9,
10] nisin- a bacteriocin produced by
Lactococcus lactis [
11‐
13] and subtilosin from
Bacillus subtilis and
B. amyloliquefaciens possess good spermicidal activity [
14].
Recombinant proteins such as heat labile enterotoxin subunit B genetically linked with hCG-β chain [
15], recombinant bonnet monkey zona pellucida (ZP1) conjμgated to diphtheria toxoid (used to immunize female baboons) [
16] and sperm specific antigen, NZ1, have been reported to prevent pregnancy [
17]. Also, various microorganisms reported to immobilize or agglutinate spermatozoa are
Escherichia coli [
18],
Chlamydia trachomatis [
19],
Mycoplasma genitalium [
20],
Ureaplasma urealyticum [
21],
Staphylococcus aureus [
22] and
Candida albicans [
23]. Hence, bacterial proteins can be explored and developed as contraceptive agents.
In this work, Staphylococcus warneri (S. warneri), isolated previously in our laboratory from the cervix of a woman with inexplicable infertility, was found to agglutinate human and mouse spermatozoa in vitro. Further, sperm agglutinating factor (SAF) was isolated and purified and was able to show complete sperm agglutination in vitro. However, as the gene responsible for sperm agglutinating activity was unknown and the production of SAF from wild type bacteria was very low, the present study was designed to identify the SAF and enhance its production by heterologous over expression and to further evaluate the efficacy of recombinant SAF as a contraceptive agent in a female mouse model.
Discussion
According to the United Nations, the world population will reach 11.2 billion by the year 2100 [
36] and contraception is the key solution to thwart this problem. Contraceptive methods to control the population are aplenty and many more are in pipeline. Condoms, intrauterine devices and oral contraceptives are successful tools available since long but they are associated with number of limitations, hence, there is an urgent need to develop safe, inexpensive and highly effective contraceptive methods. In this regard, sperm impairing agents or spermicides have come into light. Any sperm impairing agent, which immediately and irreversibly agglutinates or immobilizes the spermatozoa, not affecting developing foetus, non-irritating to penile and vaginal mucosa, non-toxic and not absorbed systemically could be a boon to field of contraception.
Various pathogens have been isolated from the semen of infertile and fertile patients interacting with the spermatozoa in terms of their agglutination and morphologic alterations [
18‐
23]. These pathogens are causative agents of genitourinary infections and affect the functioning of spermatozoa by reducing their motility and damaging their structure [
37]. Moretti et al. (2009) reported that
E. coli elicits detrimental effect on spermatozoa such as swelling of mid-piece and tail invagination [
38]. Also, they suggested the binding of
E. coli on spermatozoa and its subsequent destruction in a two-step process [
39]. Paulson and Polakoski, (1977) proposed a mechanism of sperm immobilization by
E. coli and a factor excreted by
E. coli that immobilized spermatozoa without agglutinating it [
40]. Similarly, Diemer et al., (1996) reported that binding of
E. coli results in inhibition of sperm motility via sperm agglutination [
37]. Mannose has been found to interfere with binding of
E. coli to spermatozoa but exact mechanisms of bacterial and sperm interactions have not been identified [
41].
S. aureus has also been found as predominant flora in semen of infertile men and causes infertility by reducing sperm motility [
42]. Emokpae et al. (2009) found
S. aureus as major contributor in seminal infections [
43]. Ohri and Prabha (2005) reported
S. aureus to cause sperm agglutination and an unknown protein as sperm agglutinating factor from this bacterium has been proposed responsible for this phenomenon [
44].
Role of microorganisms in impairing sperm motility is well known; hence microorganisms from the cervix of a woman with unexplained infertility were obtained and screened for sperm agglutinating activity in vitro. Clinical isolates showing positive sperm agglutinating activity were identified by MALDI.
S. warneri was the organism impairing sperm function and is a close homologue of
S. aureus, reported as sperm agglutinating agent [
45].
S. warneri was evaluated for sperm agglutinating activity and it was found that whole culture and bacterial cells washed with saline were able to agglutinate the sperm but culture supernatant could not. Sperm agglutination occurred in head-head, head-tail and tail-tail orientations that shows the presence of receptors on whole sperm. Ribonucleotide-diphosphate reductase-α sub unit gene was identified by creating shotgun genomic library that was further over expressed and purified.
Motility, acrosome status and morphology are the main parameters to determine fertilization potential of spermatozoa. Any agent interferes with any of these parameters could be exploited as contraceptive. Therefore, all these three parameters were studied by incubating sperm with rSAF and it agglutinated sperm resulting in immobilization in vitro
. Complete arrest of sperm motility has been reported within 20s using 100 μg SAF as compared to 400 μg nisin [
12] and 1 mg magainin-A indicating higher efficiency of SAF [
46]. Interestingly, Kaur and Prabha reported the irreversible effect of SAF on sperm motility as spermatozoa incubated with SAF remained immotile even after removing SAF from the reaction [
47]. This showed the irreversibility of binding and possible cytotoxicity exerted by SAF. Moreover, incubation with SAF led to the complete loss of sperm viability at high concentrations within 20s showing the efficacy and rapidity of SAF mediated sperm damage. Interestingly, effects of SAF can be neutralized by anti-SAF antiserum to inverse the infertility. Kaur et al. (2013) raised the anti-SAF antibodies and found that in the presence of antibodies, sperm agglutination was blocked by inhibiting the binding of SAF to spermatozoa thus leading to conception in mouse model [
48].
Motility is the most important character of spermatozoa required for the fertilization as immotile spermatozoa fail to meet oocyte and fertilization is inhibited. ATP provided by mitochondrian is required for sperm motility to power spermatozoa to the site of fertilization [
49]. Cation dependent ATPases are responsible for the flagellar contractile processes and active transport [
50]. Sperm moves due to ATP hydrolysis catalyzed by dyenin ATPase that is an Mg
2+dependent enzyme located on axoneme [
51]. There is a direct correlation between the sliding velocity and the quantity of dyenin arm present on the axoneme [
52]. Therefore, inhibition of Mg
2+dependent ATPase is an important parameter while studying sperm function as it is the primary sperm motility regulatory step. Hence, effect of rSAF on Mg
2+dependent ATPase activity was analyzed and the results showed that rSAF acted as potent inhibitor of enzyme and decreased the activity in a concentration dependent manner. This inhibition of sperm Mg
2+ dependent ATPase could be implicated as one of the mechanisms of impairing sperm motility by rSAF.
Premature acrosome reaction and acrosome reaction failure are crucial aspects of sperm function and are considered as important causes of infertility. The acrosome reaction is a receptor mediated exocytic process that involves outer acrosomal membrane and the sperm plasma membrane resulting in release of acrosomal enzymes required for fertilization [
53]. The sperm with intact acrosome reaching the egg is required for fertilization that undergoes induced acrosome reaction on the surface of the zona pellucida [
54]. Spermatozoa bind to zona pellucida and oocyte along with its surrounding cells release progesterone resulting in induction of acrosome reaction. Spermatozoa in cervix/vagina losing their acrosome prematurely lose their fertilization ability resulting in infertility [
55]. Kaur et al. reported the inducing effect of SAF on premature AR and apoptosis in spermatozoa [
56]. Therefore, acrosome status was analysed by incubating spermatozoa and rSAF that resulted in significantly higher rate of acrosome reaction and was comparable to CaI (positive control) when observed by fluorescent microscopy, proving its involvement in sperm damage. rSAF was found to induce premature AR in spermatozoa thus, decreasing its fertilization potential.
To understand the interaction between rSAF and spermatozoa, binding studies were carried out. When FITC labelled rSAF was incubated with human and mouse spermatozoa, fluorescence was observed over the entire spermatozoa indicating that receptors for rSAF are present on whole body i.e. sperm head, neck and tail. The uniform distribution of receptor on head, tail and body of spermatozoa resulted in mixed type of agglutination (head-tail, tail-tail and head-head). Further, intravaginal inoculation of rSAF in mice resulted in blockage of fertility was evident by absence of pregnancy related changes when observed apparently and histologically. Subsequent to assessing the contraceptive efficacy of the rSAF its toxicological impacts were likewise considered and rSAF was found to cause no toxicological effects at the contraceptive dose.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.