Contraceptive effect of sperm-agglutinating factor isolated from Staphylococcus aureus in mouse

Authors


Dr V Prabha, Department of Microbiology, Panjab University, Chandigarh-160014, India. Email satishvijay11@yahoo.com

Abstract

Objective  To investigate the contraceptive effect of sperm-agglutinating factor (SAF) isolated from Staphylococcus aureus in mouse.

Design  Experimental study performed with LACA mice obtained from the Central Animal House, Panjab University, Chandigarh, India.

Setting In vivo studies conducted in the Department of Microbiology, Panjab University, Chandigarh, India.

Population  Sixty female and 18 male mice were used for the studies.

Methods  Mice sperm–S. aureus agglutination, scanning electron microscopy (SEM), in vivo studies in mice.

Main outcome measure  Contraceptive efficacy of SAF.

Results S. aureus readily adhered to and agglutinated mouse spermatozoa. By SEM, S. aureus adherence was observed on sperm head as well as sperm tail. SAF was found to be causing 100% fertility inhibition in mouse with no effect on vaginal epithelium.

Conclusions  Sperm-agglutinating factor has been found to have a potential as a contraceptive agent.

Introduction

Vaginal contraceptive products have been available for many years and usually contain the membrane surfactant nonoxynol-9 (N-9) as one of the main ingredients.1 However, the major drawback of using N-9 or other surfactants is their detergent-type cytotoxic effect on vaginal cells.2 Besides, N-9 is also known to inactivate lactobacilli, which form the normal flora in the vaginal tissues. Disturbance of the vaginal microflora can lead to vaginal infections which in turn increase the chance of sexually transmitted infections.3 Therefore, development of vaginal contraceptive that lacks membrane toxicity will offer a significant clinical advantage over the currently marketed detergent-type spermicides.4 As an alternative, various other substances blocking or retarding the motility of spermatozoa are being examined to be useful as successful vaginal contraceptive devices, e.g. lysenin, immotilin, nisin and magainin etc. Microorganisms are also known to inhibit sperm motility either directly by agglutinating the spermatozoa or excreting extracellular products that immobilise the sperms. The influence of different uropathogenic microorganisms such as Escherichia coli, Enterococcus, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Candida albicans on human sperm motility has been studied in vitro by Huwe et al.5 Yin et al.6 showed that at high concentrations, Streptococcus viridans could induce sperm agglutination in vitro. Jiang and Lu7 reported that Staphylococcus aureus significantly immobilises the spermatozoa. In an earlier work performed in our laboratory, we have also isolated a sperm-agglutinating factor (SAF) from S. aureus, which agglutinates human and other mammalian spermatozoa.8 Further, in the present study, the efficacy of SAF as a contraceptive agent has been investigated.

Material and methods

Microorganism

The bacterial isolate, S. aureus, used in the present study was taken from the cervix of a woman with unexplained infertility attending the Department of Obstetrics and Gynecology, General Hospital, Sector 16, Chandigarh, India.

Collection of spermatozoa

Samples of mouse spermatozoa were collected from the cauda edidymis in minimum amount of physiological saline by the method of Frank.9 The morphology of the sperms including normal head and motility of sperms was observed. The number of sperms was checked by counting in a haemocytometer and adjusted to 40 × 106 spermatozoa/ml.

Animals

Sexually mature and randomly bred white mice were used for the animal experiments. LACA female mice, 8–12 weeks of age were used. All animal procedures were designed to minimise pain and discomfort.10 The study protocol was approved by the institutional ethical committee for animal experimentation.

Methods

In vitro studies

Isolation of SAF SAF was isolated from S. aureus as reported earlier.8

Sperm agglutination in vitro Equal volumes of mouse spermatozoa and S. aureus culture (72 hours old) were mixed and incubated at 37°C for 2 hour, and agglutination was observed under the light microscope (Getner, Ambala, India) at ×400 magnification.

Scanning electron microscopy Scanning electron microscopy (SEM) was carried out to study the site of adherence of S. aureus to mouse spermatozoa on a Jeol Scanning Microscope (JSM-6100; Jeol JSM-6100, Tokyo, Japan). For electron microscopy, sperm–S. aureus agglutinates were mixed in 2.5% phosphate-buffered glutaraldehyde and washed twice in phosphate buffer saline (PBS). One drop of fixed and washed sample was placed on a silver-painted adhesive tape mounted on a brass stub and air dried. Gold coating of 100 A° thickness was carried out and the specimen was observed.

In vivo studies

Effect of intravaginal application of SAF on fertility in mice For this study, a total of 36 female mice and 18 male mice were used in three fertility trials. Twelve healthy female mice were divided into two groups (control and treated), four for control and eight for treated. SAF in PBS (pH 7.2, 0.05 M) at different concentrations (2.5, 5.0, 7.5 and 10.0 micrograms) was deposited by inserting 1-ml tuberculin syringe into the vagina of four female mice. All the doses were tested in duplicate. The control animals received buffer only. The animals were mated overnight immediately after application of SAF without any delay with male mice of proven fertility (two female mice with one male mouse in each cage). The next morning, the mating was confirmed by the presence of a vaginal plug, and the mated animals were separated and kept until delivery. The experiment was repeated thrice for consistency of results.

Histological studies

For histological studies, eight mice were used in total. Two groups were formed with two mice in each group. Group 1 was the control group (with buffer only) and group 2 was the test group, treated with SAF (10 micrograms). The entire experiment was repeated to confirm the results. Animals were sacrificed on 14th day of administration of SAF. The histological analysis of different reproductive organs, vagina, bicornuate uterus, ovaries and the fallopian tubes (oviduct), was carried out according to a standard procedure. Small portions of the organs from animals of both groups were dissected out, freed from fat bodies and fixed in buffered formalin for 24 hours. Tissues were dehydrated in a graded series of ethanol, cleared in xylene, infiltrated and embedded in paraffin wax at 60°C. The blocks were sectioned at 5–7 μ using a microtome. The tissue sections were stained with haematoxylin and eosin solutions, observed under a microscope using ×10 and ×40 objectives and photographed.

Vaginal irritation test For the vaginal irritation study, two female mice were given SAF (15 micrograms) once per day and two mice were taken as control which were given buffer only once per day for ten consecutive days. Animals were killed on day 11 and the reproductive tract was examined grossly and microscopically. The vaginal tissues were rapidly removed and were fixed in 10% neutral-buffered formalin. Tissues were embedded in paraffin, sectioned at 4–6 mm and stained with haematoxylin and eosin and examined under ×200 and ×400 magnification using a light microscope. The vagina was examined for epithelial ulceration, oedema, leucocyte infiltration and vascular congestion.

Systemic toxicity in mice Two groups of mice (control and treated) with two mice in each group were used to study the systemic toxicity effect. Systemic toxicity of SAF was checked in mouse by giving intravenous injection of SAF (at concentration of 15 micrograms). The control animal received phosphate buffer (pH 7.2, 0.05 M). The animals were monitored for appearance, behaviour, other toxic signs and lethality for 15 days after administration of SAF.

The total animals used in all the experiments have been tabulated in Table 1.

Table 1.  Number of animals used in the experiments
ExperimentNo. of treated miceNo. of control mice
  • *

    Experiment was repeated thrice.

  • **

    Experiment was repeated twice.

Contraceptive effect in vivo*84
Histological studies**22
Vaginal irritation test**22
Systemic toxicity**22

Results

Sperm agglutination in vitro

Agglutinated clusters of mouse spermatozoa formed when the culture was mixed with the mouse sperms in a 1:1 ratio, but maximum agglutination of 70% of total sperms (100% of the motile sperm) was observed when bacteria and sperm were mixed at a concentration of 10:1. All of the remaining 30% of sperms that did not agglutinate were immotile. The complexes displayed mixed agglutination in which SAF formed cross-links head-to-head, tail-to-head as well as tail-to-tail.

Scanning electron microscopy

SEM showed the adherence of S. aureus on both head and tail of mouse sperm. No other structural abnormality was seen in the spermatozoa (Figure 1).

Figure 1.

SEM showing the adherence of S. aureus on head and tail of mouse spermatozoa.

Contraceptive effect of SAF in vivo

The most impressive results with SAF in relation to its antifertility potential have been observed using mouse as a model. Abdominal distention was apparent in control group mice by 12–14 days of gestation, which was absent in the treated group of mice. Similarly, small strings of pearls could be palpated in control group, which was lacking in treated group. Moreover, none of the animals instilled with the SAF protein delivered pups in comparison with the control group (Figure 2). About 8–12 pups were born on an average in the control group. Pups were not used for further studies. Each time, new group of mice was used.

Figure 2.

Control mouse with pups, 21 days after the administration of buffer.

Histology

When histology of various reproductive organs, i.e. vagina, bicornuate uterus, ovaries and the fallopian tubes (oviduct) was accessed on 14th day of introducing the SAF (10 micrograms), the results showed no alteration in vaginal epithelium in the treated and control groups.

The uterus consisting of an outer layer of smooth muscle and inner cavity lined by endometrium was seen. The endometrium in the treated group was seen as a thick, highly vascular layer containing glands and stroma, while in the control group, uterus showed the cavity occupied by a large zone of decidua and the cells were seen closely packed with abundant cytoplasm and round nuclei (Figures 3 and 4).

Figure 3.

Uterus of the control group (given buffer only) mice showing the decidua formed. Arrow indicates the decidua formation.

Figure 4.

Uterus of the treated group (given SAF) mice.

Corresponding observations were seen in the ovaries also. In treated group, the stroma with a number of germinal follicles was seen, while in the control group, the additional component of corpus luteum was seen.

Vaginal irritation test

Vaginal irritation studies were performed in the mice by repeated intravaginal application of SAF at the dose of 15 micrograms for ten consecutive days. On day 11, animals were autopsied and evaluated by gross examination of the entire vaginal area as well as complete histological evaluation of the vagina. The results showed no adverse effect on the vaginal cell morphology and vaginal epithelium, indicating no damage. The experiment was repeated twice for consistency of the results.

Systemic toxicity in mice

Intravenous injection of SAF showed no changes in the appearance and behaviour even after 15 days of administration of SAF (15 micrograms).

Discussion

Spermicides currently marketed contain nonspecific, nonionic detergents with harmful adverse effects that have only recently become known.11 N-9, in particular, has been correlated with an increased incidence of urogenital infections, cervicovaginal inflammation and epithelial changes in women using this method of birth control.12 As an alternative to spermicidal products that contain harsh chemical detergents with potentially harmful adverse effects, it was proposed that sperm-reactive monoclonal antibodies might provide safer, active topical agents.13 The recombinant antisperm antibody (RASA) generated to the entire human sperm surface sets the stage for new generations of specific protein-based sperm-agglutinating/spermicidal agents that may offer improvement over current nonspecific, detergent-based products. In the present study, we also evaluated the in vivo contraceptive efficacy of SAF which agglutinates spermatozoa in vitro. Compounds that impair sperm motility in vitro are not necessarily contraceptive in vivo.14 For example, compounds such as heparin and dextrin sulphate immobilise sperm in vitro but are not contraceptive in vivo.15 For the present study, the contraceptive efficacy of SAF was evaluated using mouse as an in vivo model. The results indicated that 2.5 micrograms of SAF was sufficient to arrest sperm motility and protect against pregnancy.

For the evaluation of the safety of new vaginal contraceptive formulations, it is important to study the toxic effects resulting from repeated intravaginal application. For vaginal toxicity studies in mice, tissue irritation is usually evaluated by gross examination of the entire vaginal area as well as complete histopathologic evaluation of the vagina. In the present study, repeated vaginal administration of SAF (15 micrograms/animal/day) for ten consecutive days caused no adverse effect on the vaginal cell morphology and vaginal epithelium, indicating no damage. These findings suggest SAF as a promising candidate in the development of contraceptive.

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