1 Introduction

2 Sites of post-testicular, epididymal contra-ception

There are three major sites of action of an epididymal antifertility agent^[6]: (i) on peritubular muscle (hastening sperm transport leading to ejaculation of young, immature spermatozoa), (ii) on the epithelium (altering the composition of epididymal fluid necessary for maturation and storage) and (iii) on the spermatozoa (attacking their characteristic enzymes).

2.1 Action on peritubular muscle activity

By removing sympathetic innervation from the distal epididymis, normal sperm transport through the epididymis is affected[8] and infertility (azoospermia) can result as sperm accumulate in the epididymis. However, this is a form of chemical vasectomy that suffers all the problems associated with the surgical technique (irreversibility, ductal rupture, antibody production). Research on shortening sperm transit times remains to be done.

2.2 Action on the epithelium

2.2.1 Inhibiting epididymal α-glucosidase

The neutral form of glucosidase is secreted by the human epididymis^[9] and is found at a microvillous localization also found in the rat^[10]. The fertility of male rats was therefore studied after epididymal glucosidase was inhibited by castanospermine, by mating them to females and counting the number of embryos present on day 12 post coitum^[11]. This inhibitor abolishes enzyme activity within 2 days of release from abdominal mini-osmotic pumps and fertility declined to 60% of controls between 7 and 9 days of treatment, but was regained over the next 5 days (Figure 1a). Since mating was permitted every 3 days, effects on sperm storage could not be evaluated. In subsequent experiments, mating was permitted after 7 and 9 days, to deplete sperm that had matured before treatment, but not again until 25, 27, and 30 days after insertion of pumps delivering drug for 28 days. Again a decline in fertility (to 50% of controls) was observed by the second mating, but there was no further reduction in fertility, which increased by day 25 and later to values found in the control animals (Figure 1b).

In a third study an attempt was made to retain sperm in the epididymis by ligating the efferent ducts and allowing them to remain in the distal cauda epididymidis until ejaculation. This design permits investigation of sperm that had only ever been through, and stored in, a glucosidase-free environment. A significant decline (to 60% controls) was achieved by castanospermine treatment on day 29 (14 days after ligation of the efferent ducts) compared to ligated controls (Figure 1c). This suggests that glucosidase may be involved in the storage of sperm in the epididymis, but the effect on fertility is small. This approach does not look like a promising lead for a contraceptive.

Figure 1. Fertility of male rats (number of embryos per corpus luteum, expressed as a percentage) when inserted with osmotic mini-pumps containing the glucosidase inhibitor castanospermine (s) for (a) 14 days with matings every 2 or 3 days starting from day 7; (b) 28 days pump with matings after 16 day abstention, after initial sperm depletion; (c) 14 days before and after efferent duct ligation on day 15 with matings on day 29. Fertility partially declines after initial sperm reserves are emptied but returns in (a) and (b), but remains depressed (*) after sperm have matured and been stored in a glucosidase-free epididymis in (c). Controls(●) were treated the same way but castanospermine was replaced by phosphate-buffered saline. Bars indicate duration of castanospermine release. Data from 11.

2.2.2 Lowering the epididymal carnitine content

The high concentration of carnitine in epididymal fluid may reflect a high carnitine requirement of epididymal spermatozoa. As it has been claimed to be involved in the acquisition of sperm motility^[12], a reduction in the carnitine concentration in the epididymal lumen should affect sperm motility, and as a consequence, fertility after natural mating. Carnitine is concentrated by the epididymal epithelium from the bloodstream^[13] by a carrier that recognizes both ends of the molecule^[14]. There are no known inhibitors of this pump but the low K_m of carnitine transport (around blood levels) implies that reducing the concentration circulating would lower the epididymal content. As administration of pivalic acid via the drinking water decreases serum carnitine in rats, by increasing urinary excretion of pivaloylcarnitine, this was employed to decrease epididymal carnitine in rats and hamsters of proven fertility. In rats, the carnitine content of serum, of epididymal tissues and of cauda epididymidal fluid, was lowered by 50-75% on addition of sodium pivalate (20 mmol/L) to the drinking water over a 5-week period (Figure 2). Despite this, there was no decrease in the fertility of the rats or of the motility of spermatozoa from four regions of the epididymis when measured by computer-aided sperm motion analysis (CASA: 15). However, the carnitine content of distal caudal epididymidal spermatozoa was not lowered by this treatment and further experiments with 60 mmol/L pivalate did not lower fertility.

Figure 2. Carnitine concentrations and fertility of male rats fed sodium pivalate to increase urinary excretion of carnitine. (a) Carnitine concentrations in blood (○, mmol/L) and in epididymal fluid (q, μmol/mg protein) decrease by 70% in one and two weeks. (b) Carnitine in epididymal tissue (μmol/mg protein) decreases in two weeks by 80% in epididymal regions 3 (●), 4 (s), 5 (■), and 6 (¯) or by 60% (region 7, ▲) and 40% (region 8). (c) Fertility (number of embryos per corpus luteum, expressed as a percentage) in control (q) or pivalate-fed rats (○) are not different. Data from 15.

A more marked reduction in epididymal carnitine (to 26 % of controls) was found in hamsters treated with pivalic acid or sodium pivalate for up to a month^[16] and their fertility was assessed by intra-uterine insemination of 3×10⁶ distal cauda epididymidal spermatozoa and examining ova collected from the female tract 24-40 h later. Again, there was no difference in the percentage of males that was fertile, but the percentage of eggs fertilised was lower when sperm were taken from the pivalate-handled hamsters (13%) than from controls (44%), despite the lack of changes in sperm motility.

In neither species was total infertility induced in animals whose epididymides were partially depleted of carnitine, so this approach to contraception did not look promising.

2.2.3 Immunological sequestration of specific proteins

Contraception is more likely when the epididymal secretions targeted are limiting and this may be the case for specific proteins that are taken up by spermatozoa as they mature or are stored. A pre-albumin specific protein (PES, protein D/E), has been implicated in sperm-egg binding and fusion with the vitellus and used as an immunogen for males. In rats, active immunization against protein D/E induces antibodies against the protein within days and reduced fertility within months^[17,18]. Immunization against PES reduced in vivo fertility in weeks and epididymal sperm motility and zona binding were reduced in the immunized animals^[19] but there was no full recovery of fertility 6 months after injections began. In a related study, rats immunized against the ovine PES homologue remained fertile despite production of antibodies^[20]. By contrast, active immunization of rams against the ovine protein did lead to reduced motility of sperm in the ejaculate during the time that specific antibodies were detectable in the serum and semen. Fertility was normal when sperm motility had recovered after antibodies were no longer detectable, although it was not checked during the period of depressed motility^[21]. As the equivalent protein in man^[22] is only loosely bound to the sperm surface^[23], it is unlikely that such immunization would achieve contraception in man.

In the hamster, antibodies formed after active immunization against the epididymal-specific protein P26h were found on the surface of spermatozoa within the epididymal lumen^[24]. The immunized hamsters displayed no testicular damage and the treatment resulted in the complete lack of fertilised ova recovered the day after mating. Because no sperm were found on the zona pellucida or in the perivitelline space of the unfertilised eggs, fertilisation failure probably stemmed from inhibition of sperm-zona binding. This implies that this method is capable of generating sufficient antibodies in the lumen to neutralise all the proteins involved in fertilisation. These promising results have led to clinical trials being planned for immunization against the human equivalent of this protein, P34H, the amount of which on sperm is related to the fertility of men^[25,26]. 

2.3 Interfering with sperm function

2.3.1 Interfering with sperm membrane ion channels

Adult homozygous c-ros tyrosine kinase knockout male mice are infertile by natural mating and the only phenotypic abnormality is in the epididymis that fails to develop an initial segment^[27]. They are a unique model to study the role of the epididymal epithelium in regulation of sperm function and present a possible point for contraceptive attack. The infertility in vivo contrasts with ability of spermatozoa from the cauda epididymides to fertilise eggs in vitro, which suggests that sperm suffer a defect in transport in the female. Fewer sperm are indeed recovered from the oviducts of the females mated to homozygous males although computer-aided analysis of sperm motion parameters revealed no differences between the knock-out and wild type animals^[5]. An obvious phenotype of the spermatozoa from the homozygous knockout male was the high percentage of flagella displaying pronounced bending (angulation) at the position of the cytoplasmic droplet, a phenomenon not found in the wild type or heterozygous control sperm.

Angulation was found to some extent within the epididymis of the knockout mice but was exaggerated on dilution in normal medium. The bent shape of the knockout sperm can be mimicked in wild type sperm by addition of agents that hinder cell volume regulation (CH Yeung & TG Cooper, unpublished data) which suggests that the failure of knockout sperm to osmoregulate may underlie the infertility of the animals. Although there are no infertility syndromes defined by the presence of sperm with coiled tails, inducing coiled tails in human sperm would prevent their access to the egg. The defect in the c-ros knockout males must arise from some incomplete or abnormal interaction between maturing sperm and epididymal factors as a consequence of the lack of a differentiated initial segment. Disrupting the production or function of these factors should have contraceptive potential and is currently under study.

2.3.2 Inhibiting sperm glycolysis

Ornidazole is a nitroimidazole compounds that rapidly induce infertility in male rats without an effect on the testis^[28] and has an effect on the motility of cauda epididymidal spermatozoa. To define the mode of action of this drug, ornidazole was administered daily to male rats at 400 mg/kg for 2 or 3 weeks. Infertility was rapidly induced with some males being subfertile after 3 days, most by 6 and all animals being infertile by 14 days, in sharp contrast to vehicle-fed controls which exhibited 80-95% fertility. When fed at half of this dose male rats never became infertile but subfertility persisted up to 3 weeks of daily feeding, suggesting that a critical dose of the drug or its metabolite had to be produced to affect all the spermatozoa. After withdrawal of the drug, fertility rapidly returned within 7 days to control levels (Figure 3).

Figure 3. Fertility of male rats (number of embryos per corpus luteum, expressed as a percentage) when fed ornidazole at 400 mg/(kg·d) for 2 weeks (s), 200 mg/(kg·d) for 3 weeks (●), vehicle alone for 3 weeks (■) or 400 mg/(kg·d) for 1 week followed by vehicle for 1 week (¯). Data from 29.

The rapid action implies a post-testicular effect, but since no changes in epididymal secretions (glucosidase, carnitine, GPC) were observed, a direct action on epididymal spermatozoa was concluded^[29]. The motility of sperm from the distal epididymis of ornidazole-fed rats, assessed by CASA in vitro, was indeed reduced and significantly fewer sperm were recovered from the oviduct and penetrated the cumulus^[30]. None of the eggs was fertilised and sperm were not seen on the zona pellucida or in the perivitelline space. Two glycolytic enzymes (triosephosphate isomerase and glyceraldehyde 3-phosphate dehydro-genase) were inhibited in sperm from the infertile rats but there was no reduction in the ability of the sperm to undergo capacitation or the acrosome reaction in vitro^[31].

By feeding several ornidazole analogues to male rats at doses equivalent to an antifertility dose of ornidazole, the chlorinated side chain was shown to be indispensable for infertility action^[32]. Ornidazole, radiolabelled in the side chain, fed to male rats underwent extensive dechlorination, as free ³⁶Cl, as well unconverted ornidazole was found in the urine. The presence of 3-[³⁶Cl] chlorolactic acid, the oxidation product of 3-[³⁶Cl] chlorolactaldehyde^[33], suggests strongly that ornidazole is metabolized to 3-chlorolactaldehyde, a known inhibitor of GAPDH produced by other antifertility compounds (Figure 4). As a result, ATP production from glucose is limited, sperm velocity is reduced and fertilisation in vivo prevented.

Figure 4. Suggested metabolism of chloro-compounds (left, amino-compounds; right, chloro-sugars; bottom, glycerol derivatives) to 3-chlorolactaldehyde (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), based 34, 35, 33, 36. Abbreviations: CHOP, 3-chloro-1-hydroxypropanone; GDH, glycerol dehydrogenase; MAO, monoamine oxidase; TPI, triose phosphate isomerase.

The inhibition of sperm glycolysis is specific since high dose of α-chlorohydrin (90 mg·kg^-1·d^-1) for 2 to 10 days does not decrease glycolytic activity of several other tissues^[37]. Sperm enzymes are unable to distinguish between chlorinated and phosphorylated compounds^[38] and the difference in sensitivity reflects differences in the inhibited enzymes. Sperm glyceraldehyde 3-phosphate dehydrogenase is not cytosolic, as in somatic cells^[39], is a larger isoenzyme^[40] and is physically linked to the fibrous sheath^[41] that surrounds the axoneme. For sperm, glycolysis may be more important than respiration in generating ATP adjacent to the site of utilisation by dynein ATPase. Just as known antifertility agents liberate (S)-3-chlorolactaldehyde from chlorohydrins, amino compounds or chlorosugars, so future contraceptives may be based on chloro-analogues of dihydroxyacetone phosphate^[42] or dichloro-dideoxyfructose^[35] (Figure 4). Preliminary studies (W Bone & TG Cooper, unpublished) have indicated that sperm motility and glycolytic enzymes are inhibited by these compounds (Figure 5).

Figure 5. The in vitro inhibition by increasing concentrations of α-chlorohydrin (ACH) and chlorohydroxypropanone (CHOP) (abscissa) of motility (a) VCL (curvi-linear velocity, μm/s), (b) VSL (straight-line velocity, μm/s) and glycolytic enzymes, (c) GAPDH (glyceraldehyde 3-phosphate dehydrogenase, mU/10⁸sperm), (d) TPI (triosephosphate isomerase, mU/10⁸ sperm), of ejaculated human spermatozoa. Data are all expressed as percentages of drug-free control values (ordinate) and given as mean±SEM (n=4). Unpublished data from W Bone and TG Cooper.

2.3.3 Displacing sperm proteins

3 Conclusions

4 Acknowledgements

References

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