to post-testicular contraception
G Cooper, CH Yeung
of Reproductive Medicine of the University, Münster, Germany
Asian J Androl 1999 Jun; 1: 29-36
Keywords: epididymis; contraception; antifertility; male infertility; animal models
AbstractThe induction of infertility in males of several species through epididymal interference is more difficult to achieve by reduction of the amounts of epididymal secretions (eg α-glucosidase, L-carnitine) or immunological interference with secreted proteins (eg D/E, P34H, P26h) than by direct actions of drugs on sperm function (eg inhibition of glyceraldehyde 3-phosphate dehydrogenase by chloro-compounds). The latter approach holds promise for mankind as human sperm are susceptible to glycolytic inhibition. Future contraceptive developments may arise from production of targeted inhibitors, research on the displacement of sperm proteins in the epididymis and interference with sperm plasma membrane ion channels.
1 IntroductionThe burgeoning world population and the associated risk of damage both to the planet and human health have led to calls for increased availability of family planning and also for men to share in this responsibility. In contrast to a variety of services for women, men have only three choices: withdrawal, vasectomy and the condom, with their associated problems of low efficacy and/or irreversibility. While hormonal contraception is the most extensively studied and becoming acceptably efficacious[2,3], it suffers from a relatively long time to azoospermia. In this review we survey the post-testicular, epididymal approach that has the benefits of (i) almost immediate effectiveness, (ii) ready reversibility and (iii) avoidance of psychological or endocrine impairment of libido. As sperm are matured and stored in the epididymis, under the influence of epididymal secretions[4,5], contraceptive agents could influence spermatozoa indirectly, through disruption of epididymal epithelial cell function or act directly on them. The notion that contraception could be based on action in the epididymis is supported by several cases of natural infertility in domestic species and a knock-out mouse model in which failure of epididymal development is associated with infertility.
2 Sites of post-testicular, epididymal contra-ception
are three major sites of action of an epididymal antifertility agent:
(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).
Action on peritubular muscle activity
removing sympathetic innervation from the distal epididymis, normal sperm
transport through the epididymis is affected 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.
Action on the epithelium
Inhibiting epididymal α-glucosidase
The neutral form of glucosidase is secreted by the human epididymis and is found at a microvillous localization also found in the rat. 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. 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).
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.
Lowering the epididymal carnitine content
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, 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 by a carrier
that recognizes both ends of the molecule.
There are no known inhibitors of this pump but the low Km
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
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 (●),
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 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.
neither species was total infertility induced in animals whose epididymides
were partially depleted of carnitine, so this approach to contraception
did not look promising.
Immunological sequestration of specific proteins
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 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. 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. As the equivalent
protein in man is only loosely bound to the sperm surface,
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. 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].
Interfering with sperm function
Interfering with sperm membrane ion channels
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. 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.
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.
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.
Inhibiting sperm glycolysis
Ornidazole is a nitroimidazole compounds that rapidly induce infertility in male rats without an effect on the testis 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.
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. 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.
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.
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. 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, 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. Sperm enzymes are unable to distinguish between chlorinated and phosphorylated compounds and the difference in sensitivity reflects differences in the inhibited enzymes. Sperm glyceraldehyde 3-phosphate dehydrogenase is not cytosolic, as in somatic cells, is a larger isoenzyme and is physically linked to the fibrous sheath 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 or dichloro-dideoxyfructose (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.
Displacing sperm proteins
3 ConclusionsFrom the data obtained so far from animal models, the induction of infertility in males of several species has been achieved by direct actions of drugs on sperm function (glycolytic inhibition) rather than indirect actions mediated by modulating epididymal secretions (glucosidase, carnitine, sperm-coating proteins). The indirect method has brought little success because epididymal secretions are not produced at limiting rates but accumulated at high concentrations within the epididymal canal by normal epididymal functioning that ensures that all spermatozoa are affected by its secretions. Epididymal concentrations may have to be reduced enormously for an effect of their withdrawal to be observed, and this may not be achievable rapidly or at all. On the other hand, the infertile c-ros knock-out mouse model indicates that an epididymal approach is plausible, providing the as yet unknown epididymal factors controlling sperm cell function can be identified and targeted. The action of ornidazole and other pro-drugs in inducing rapid, reversible infertility in males is currently an attractive post-testicular approach and is due to production of (S)-3-chlorolactaldehyde, a known inhibitor of sperm-specific isoenzyme GAPDH and possibly the displacement of a protein involved in other aspects of fertilisation. Further research with these compounds should combine these features with directed accumulation of the agents around the sperm in the epididymis. This would increase therapeutic ratios both by reducing the doses required for adequate contraception and lowering the chances of side effects.
WHO Male Contraception: 1993 and Beyond. In: PFA Van Look and G Pérez-Palacios,
editors. Contraceptive Research and Development, Delhi: Oxford University
Press, 1994. p 121-34.
Correspondence to TG Cooper, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany.
Tel: +49-251-835 6449 Fax: +49-251-835 6093
Received 1999-03-15 Accepted 1999-05-09