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Biol Reprod 2004, 10.1095/biolreprod.104.031252
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BIOLOGY OF REPRODUCTION 71, 1405–1411 (2004)
DOI: 10.1095/biolreprod.104.031252
© 2004 by the Society for the Study of Reproduction, Inc.

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Sperm Survival Versus Degradation in the Mammalian Epididymis: A Hypothesis1

Roy Jones2

Laboratory of Molecular Signalling, The Babraham Institute, Cambridge CB2 4AT, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ARE THERE ANY CIRCUMSTANCES...
 HYPOTHESIS
 CONCLUSIONS
 REFERENCES
 
A long-standing problem in epididymal physiology is the fate of unejaculated spermatozoa in the cauda epididymidis under conditions such as congenital absence of the vas deferens, long-term vasectomy, or castration. There is no convincing evidence for significant absorption of spermatozoa, defective or otherwise, by spermiophagy or dissolution in the epididymis of normal animals. Spermiophagy by epithelial cells or intraluminal macrophages may take place if the duct ruptures and granulomas form (e.g., after experimental ligation), although there is no quantitative information on the rate of sperm removal by this means. In one animal model (the rabbit), the epididymis is unusually resistant to granuloma formation and has provided unique insights into a phenomenon that is suggested to be present in all species. Spermatozoa retained in the rabbit cauda epididymidis by placing ligatures on the vas deferens and corpus epididymidis degenerate after several weeks but do not decrease significantly in numbers. After castration, however, they die very rapidly and >90% disappear. It is hypothesized that, in the normal androgen-maintained epididymis, degradative pathways are present in the luminal fluid that are constitutively inhibited by survival signals emanating from the epithelium. In the absence of androgen, the intraluminal mileau changes and death signals predominate that activate degradative pathways via the ubiquitin-proteasome system, DNAses, etc., to mediate dissolution of sperm organelles and nucleoprotein. It is suggested that the latter condition is the default situation and is only prevented by the stimulatory action of androgens on the epididymal epithelium.

aging, death signals, epididymis, male reproductive tract, proteolysis, sperm, sperm degradation, survival signals, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ARE THERE ANY CIRCUMSTANCES...
 HYPOTHESIS
 CONCLUSIONS
 REFERENCES
 
The mammalian epididymis has two principal functions. First, it creates a unique microenvironment within the lumen of the duct that helps transform immotile, immature testicular spermatozoa into fully fertile competent cells, and second, it stores fertile spermatozoa in a viable state within the cauda epididymidis/vas deferens regions until they are ejaculated. The phenomenon of sperm maturation in the epididymis is well documented in the literature and considerable effort has been devoted over many years to identifying the processes involved [1–3]. In particular, much attention has centered on the morphology and secretory activity of the epithelial cells lining the duct, the composition of the luminal fluid and its influence on the contained spermatozoa, as well as detailed descriptions of remodeling processes to the sperm plasma membrane, nucleoprotein, and acrosome that correlate with acquisition of fertilizing capacity [4–6]. Although doubts occasionally arise about the extent of sperm maturation in the human epididymis [7–9], there is sufficient evidence to indicate that it takes place and that similar mechanisms prevail as those described in other mammalian species [10, 11].

There is also much antique literature describing the prolonged survival of spermatozoa in the cauda epididymidis of normal animals [1, 12–17]. Estimates vary from several days to weeks, depending on the species. In scrotal mammals, the combination of a unique luminal milieu and lower temperatures (30–32°C) are thought to be major contributors to sperm survival [18, 19]. It has even been suggested that the need to store spermatozoa prior to ejaculation has been a major selective force in the evolution of the scrotum [20]. Remarkably, if spermatozoa are removed from the cauda epididymidis and incubated at 32°C in vitro, their fertility and viability is measured in hours rather than days.

An equally long-standing suggestion is that the epididymis functions as a quality-control organ to prevent misshapen, genetically abnormal or infertile spermatozoa from entering the ejaculate. The origins of this hypothesis can be traced back to early observations in the 1930s and 1950s by Simeone and Young [21] and Ortavant [22] that were subsequently extended by several different workers in the 1960s in [23–26]. In the bull, goat, dog, and cat epididymis, Glover [23] reported a higher incidence of decapitate and eosinophilic spermatozoa in the proximal corpus than in the cauda, implying that they must have been removed at some point, presumably in the distal corpus. Proposed mechanisms for their adsorption have ranged from dissolution within the duct lumen [25] to spermiophagy by macrophages [27, 28] and principal cells that line the duct [26, 29–31]. In the 1970s, however, extensive work on estimates of daily sperm production using newly developed techniques of quantitative testicular histology and cannulation of the rete testis and vas deferens failed to substantiate the hypothesis that, in the normal animal, significant numbers of spermatozoa disappeared during epididymal passage [32, 33]. Furthermore, extensive histological and ultrastructural analysis of the normal epididymal epithelium demonstrated that phagocytosis of spermatozoa was a rare event and incompatible with disposal of the millions of spermatozoa produced daily by the testis.

Recently, the concept that the epididymis acts as a quality-control organ to remove defective spermatozoa before ejaculation was resurrected by Sutovsky et al. [34] on the basis that a small proportion of spermatozoa in the corpus epididymidis of the bull were ubiquinated and cultured principal cells are capable of spermiophagy. The deficiencies in this evidence have been summarized by Cooper et al. [35]. Suffice it to say that, despite these claims, the balance of evidence does not support the view that, in the normal animal, the epididymis routinely absorbs significant numbers of defective or dead spermatozoa. If a sperm quality-control system is present in the epididymis, then it is more subtle than phagocytosis of dead or disintegrating spermatozoa.


   ARE THERE ANY CIRCUMSTANCES WHERE SPERMATOZOA ARE ABSORBED IN THE EPIDIDYMIS?
TOP
 ABSTRACT
 INTRODUCTION
 ARE THERE ANY CIRCUMSTANCES...
HYPOTHESIS
 CONCLUSIONS
 REFERENCES
 
The foregoing discussion refers to the situation in the normal mature animal. That is, where the epididymal duct is fully patent and receives a constant supply of androgens directly via testicular fluid and indirectly from the blood (the situation in senescent animals may be different, as other factors related to general aging, e.g., reduced pituitary function, lower androgen profile, are influential). There are conditions, however, where loss of spermatozoa from the epididymis has been reported and it is relevant to consider the credibility of this evidence and the possible mechanisms involved.

The first condition is obstruction of the epididymal duct or vas deferens brought about by experimental ligation or congenital absence of the cauda epididymidis and vas deferens. The latter condition (known as CAVD) is well known clinically in man as it is an obvious cause of infertility and is associated with mutations in the cystic fibrosis gene [36]. Testicular biopsies on these patients has revealed active spermatogenesis in the majority of cases [37], raising the question of what happens to spermatozoa in the excurrent ducts. Little information is available on this problem aside from isolated observations that large numbers of defective (i.e., misshapen) spermatozoa are present in the epididymis [38, 39]. Presumably over many years, a balance is reached between sperm production in the testis and a slow chronic rate of spermiophagy in those regions of the epididymis that are still present (see later). Vasectomy cannot be equated with CAVD, as in the latter case, the obstruction is present from birth and the testis and epididymis will have had time to accommodate gradually to the condition over a long period of time. Consequently, antisperm antibodies are rarely present in the serum from CAVD patients. Following vasectomy, however, an obstruction is suddenly imposed on a previously patent duct and an inflammatory reaction frequently ensues as sudden backpressure from accumulating spermatozoa causes the duct to swell and eventually burst, leading to formation of sperm granulomas. Not surprisingly, >80% of vasectomized men have antisperm antibodies in the peripheral blood within a year [40]. In a survey on the effects of chronic vasectomy in laboratory animals (hamster, rat, and monkey), Bedford [41] observed rapid formation of sperm granulomas in nearly all cases, either at the site of ligation on the vas deferens or in the cauda epididymidis. It was concluded that "their [sperm] disposal depends on ingestion by leucocytes, which invade the duct first via the granulomata and then migrate within the tract, and, particularly within the terminal vasal cysts, by epithelioid cells which bound the periphery of such sites." Unfortunately, quantitative estimates of the number of spermatozoa disposed of in this way are difficult to make.

In this respect, the rabbit has proved a better experimental model as the vas deferens and cauda epididymidis are very distensible and, following vasectomy, can accommodate considerable backpressure from accumulated fluid and spermatozoa without rupturing [41, 42]. Moore and Bedford [43] observed a linear increase in the number of spermatozoa in the epididymis of rabbits vasectomized for up to 6 mo (in these investigations, granulomas did not form on the vas deferens) that equated closely with estimates of sperm production by the testis. This argues strongly against sperm resorption or dissolution despite the fact that the majority of spermatozoa proximal to the site of ligation on the vas deferens would be dead and decapitate after 6 wk. The presence of large numbers of phagocytes in the lumen of the duct was not a reported feature in any of these studies. On this basis, therefore, it would seem that, when the duct does not rupture and granulomas do not form, massive invasion of phagocytes through the epithelium does not take place and sperm numbers do not change significantly.

It is fair to point out that, in addition to the CAVD condition, spermiophagy by epithelial cells lining the ductuli efferentes, caput epididymidis, and vas deferens has been reported on several occasions, especially following blockage of the duct by experimental ligation [28, 30, 44–48]. Once again, quantitative estimates of the rate of disposal of spermatozoa and their fragments by this means are lacking. At best, it must be low and unlikely to approach the rate of sperm production in the testis. The available evidence, therefore, indicates that, although there is a level of spermiophagy by the epididymal epithelium in response to obstruction of the duct, it cannot be a major route for disposal of the several billion spermatozoa that are normally present in the epididymis and the millions that enter it every day from the testis. Eventually, over a longer period that probably varies with the species, backpressure from the site of obstruction will reach the testis and spermatogenesis will be disrupted and reduced to a much lower than normal level.

The second situation where epididymal sperm numbers have been reported to decline is following bilateral castration. In keeping with other male accessory sex glands, the epididymis is androgen dependent and, following castration, the epithelium regresses rapidly, especially in the initial segment and proximal caput epididymidal regions, which receive an additional supply of testosterone and growth factors from testicular fluid. As a consequence, sperm maturation and survival are adversely affected and, after several weeks, only a few degenerated spermatozoa are present in the cauda epididymidis and vas deferens. A comparable situation is found in seasonal-breeding animals at the time of testicular regression, when large numbers of semicondensed spermatids and decapitate spermatozoa are present in the epididymis [49, 50]. Most of these sperm remnants are probably voided to the exterior via the vas deferens. However, a different situation arises when ligatures are placed experimentally on the vas deferens and corpus epididymidis so that spermatozoa can neither leave nor enter the cauda epididymidis (an experimental condition known as the isolated cauda) and at the same time both testes are removed. In the hamster, Lubicz-Nawrocki [51] reported that, 2 wk after castration, <5% of spermatozoa could be recovered relative to the isolated cauda in normal intact animals. No mention was made in this paper of granuloma formation, although this was later challenged by Temple-Smith and Bedford [52], who observed an incidence of 67% granuloma formation on the epididymis following castration. In the mouse, we have also observed a high incidence of granuloma formation on the vas deferens following ligation and castration (25%, 90%, and 60% after 1, 2, and 3 wk, respectively; unpublished observations). Thereafter, the granulomas decreased in size and, by 4 wk, were difficult to detect morphologically, presumably because the duct was in an advanced state of involution and most of the spermatozoa had been phagocytosed by invading macrophages.

Again in the rabbit model, the situation is different. Jones and Glover [53] reported a gradual decline in the percentage spermatocrit (packed cell volume) in the isolated cauda for the first 4 wk after castration and, by 5 wk, >95% of spermatozoa had disappeared. This was not explicable by formation of granulomas, invasion of the lumen by large numbers of leucocytes, or spermiophagy by the regressing epithelium. Unfortunately, sperm numbers were not recorded in these experiments, but in a reexamination of the phenomenon, in which the total number of spermatozoa in the isolated cauda was counted accurately by computer analysis and each animal was used as its own control, sperm disappearance after 5 wk of castration was confirmed (Table 1) and, more importantly, could be prevented by administering testosterone, thereby excluding adverse side effects of the surgical procedures [54]. The importance of using each animal as its own control is shown by the 10-fold difference in sperm numbers between individual rabbits. Studies that do not take this into account can be severely distorted by this variation. The result with the castrated testosterone-supplemented animals is also in keeping with early work by Paufler and Foote [29], who placed ligatures on the vas deferens and corpus epididymidis of rabbits and left the testes in situ. They found that the number of spermatozoa present within the isolated cauda did not decrease significantly over a 4- to 12-wk period. No mention was made of the presence of granulomas. In the presence of androgens, therefore, spermatozoa may degenerate but they are not reabsorbed to any appreciable extent or, at best, only extremely slowly.


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  		TABLE 1. Total number of spermatozoa (x 106) in the control and experimental cauda epididymidis of rabbits castrated for 3 or 5 wk ± testosterone supplementation.a

If the disappearance of spermatozoa from the isolated cauda of the rabbit after castration cannot be accounted for by granuloma formation, spermiophagy by the epididymal epithelium, or invading macrophages, what possible mechanism could account for it? One explanation is that, under these circumstances, spermatozoa undergo degradation and dissolution of some kind within the epididymal lumen. Speculations about this possibility can be found throughout the literature. Waldschmidt et al. [25] first reported DNAse activity in sperm-free plasma from the lumen of the cauda epididymidis (known as CEP) of bulls and suggested that spermatozoa are suspended in a potentially degradative milieu. Subsequent investigations confirmed the presence of acid DNAse activity and a variety of other nucleolytic enzymes [55, 56]. More significantly, in the present context, a positive reaction for deoxyribose was obtained in CEP from castrated rabbits at a time when sperm numbers were declining rapidly. Values equivalent to 0.75, 1.81, 3.58, 3.78, and 2.55 mg DNA/100 ml were measured after 1, 2, 3, 4, and 5 wk of castration, respectively, the implication being that it was derived from degenerating spermatozoa because there was no detectable reaction in CEP from normal animals or from castrated animals receiving testosterone supplementation [57]. The pH of CEP also rose rapidly following castration from its normal value of ~6.7 to >8.0 that, at the time, was explained by the release of protamines from decondensing sperm heads [58]. Because a similar increase in pH was observed after androgen withdrawal in animals with a sperm-free epididymis, a more likely explanation is the loss of function of ATP-dependent proton pumps in the apical surface of epithelial cells lining the duct [59].


   HYPOTHESIS
TOP
 ABSTRACT
 INTRODUCTION
 ARE THERE ANY CIRCUMSTANCES...
 HYPOTHESIS
CONCLUSIONS
 REFERENCES
 
Sperm survival in the cauda epididymidis is maintained by the normal functioning of the androgen-stimulated epithelium. If androgens are removed or decline substantially, e.g., after castration, mechanisms are activated that degrade and dispose of dead spermatozoa by dissolution.

In this hypothesis, sperm survival or degradation in the cauda epididymidis is a balance between survival and death signals controlled by the epididymal epithelium (Fig. 1). In computer software terminology, sperm degradation is the default situation but is prevented by the androgen-stimulated epithelium maintaining a prosurvival environment within the lumen of the duct. Although the evidence for this hypothesis rests largely on data derived from the rabbit model, the latter should not be regarded as unique or unusual. For reasons discussed earlier, it manifests a phenomenon that is present in varying degrees in all species but is difficult to demonstrate experimentally because of the general tendency of the vas deferens to form granulomas after occlusion of the duct.



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  		FIG. 1. Model of the proposed balance between survival and death signals for spermatozoa in the lumen of the cauda epididymidis

Following malfunctioning of the epididymal epithelium, one of the earliest indications of an increasingly stressful environment in the lumen is the appearance of large numbers of coiled tail and eosinophilic spermatozoa. Thereafter, they quickly become decapitate. The problem now is to dispose of the resistant sperm head, mitochondria, fibrous sheath, and outer dense fibers. Phagocytosis by the epithelium is not a viable option as the principal cells are undergoing rapid involution. One scenario is activation of proteinases and hydrolases in the luminal fluid as a result of changes in pH, ionic strength, etc., which would facilitate solubilization of many of the aforementioned organelles. Disposing of the condensed sperm head, however, is more difficult. In vitro, an effective way of decondensing sperm heads is trypsinization (to cause decapitation and dissolution of the perinuclear theca) in the presence of a reducing agent, such as dithiothreitol. A reducing environment seems important because, without it, sperm nucleoprotein decondenses very slowly [60]. It is possible that, if sufficient reducing activity was present in epididymal fluid (e.g., from glutathione), then, in the presence of active proteinases and DNAases, decondensation and degradation of sperm heads would begin and they would gradually disappear as recognizable entities over a period of days or weeks.

In support of this hypothesis, we have detected on agarose gels a ladder-like pattern of DNA fragments in luminal fluid collected from a 4-wk castrated rabbit (Fig. 2A) that is consistent with the detection of deoxyribose in CEP as mentioned earlier. The most likely source of this degraded DNA is spermatozoa. Again, in confirmation of earlier data, we have detected active DNAse activity in bull, boar, rabbit, and rat CEP when incubated with calf thymus DNA at pH 5.0 (Fig. 2B). The genomic DNA, which remained partially at the origin and partially at a band ~10 kb in size, was degraded to a range of smaller fragments, some of which accumulated ~0.2 kb. Bull, rabbit, and rat CEP contained noticeably higher DNAse activity than boar CEP. The converse was apparent at pH 7.8, where boar CEP degraded the calf thymus DNA more rapidly than the other three species. Thus, the DNAse activity in CEP is potentially active over a broad pH range.



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  		FIG. 2. Detection of DNA and DNAses in CEP. A) CEP from normal and 4-wk castrated rabbit. Note the ladder-like distribution of DNA fragments in the castrated sample. B) Degradation of calf-thymus DNA (Sigma, Poole, UK) by DNAses in CEP from bull, boar, rabbit, and rat at pH 5.0 and pH 7.8. CEP samples were adjusted to 8.0 mg/ml total protein [74]. C) Control samples containing either no enzyme or purified DNAse I (bovine pancreas; Roche, Lewes, UK). Incubation mixtures contained 2.5 µg DNA in 25 µl of 0.1 M acetate/5 mM MgSO4 buffer, pH 5.0, + 2 µl of CEP or purified enzyme. For pH 7.8, the buffer was adjusted with 5 mM Tris. Samples were incubated at 37°C for 60 min, 5 µl removed and electrophoresed on 1% agarose gels followed by staining with ethidium bromide

In addition to the capacity to degrade DNA, ubiquinated proteins are present in bull, boar, rabbit, and rat CEP as detected on Western blots with an antiubiquitin antibody (Fig. 3A). Ubiquitin is a small (8.5 kDa), highly conserved protein that binds to other proteins, marking them for degradation via the 26S proteasome system [61]. Ubiquitin has been detected immunologically in the apical cytoplasm of principal cells in the cauda epididymidis of the bull and rat and on supposedly defective spermatozoa in the lumen [34, 62]. The 20S proteasome complex is also present in bull, rabbit, and rat CEP when Western blots are probed with an antibody to an internal peptide in the human HC2 subunit (Fig. 3B). A strong reaction was detected in bull CEP at the expected size of ~32 kDa (the HC2 is ~29 kDa) with weaker reactions in rabbit and rat CEP at ~30 kDa. No reaction was detected in boar CEP; this may reflect a problem of antibody cross-reactivity between species. We have not investigated the presence of the 20S proteasome complex in bull, boar, rabbit, and rat spermatozoa, although it has been reported in ascidian, mouse, and human spermatozoa [63–65]. It is possible, therefore, that, in addition to their counterparts in the CEP, endogenous proteasomes, acrosomal proteases, such as acrosin and DNAses [66, 67], contribute to sperm degradation. It is worth noting in passing that the optimal pH for autoactivation of proacrosin to acrosin is ~8.2 and that, besides ubiquitin, other CEP proteins bind to and mark nonviable sperm [68]. The identity and function of these proteins is not known. However, implications that spermatozoa undergo classic apoptosis or programmed cell death should be treated with caution, as there is little experimental evidence to support this view [69]. Apoptotic markers, such as fas and caspase antigens, on human sperm [70] may well be leftovers from earlier remodeling processes that took place during meiosis and spermiogenesis. Nonetheless, it is conceivable that leakage of cytochrome c from the mitochondria of degenerating spermatozoa could bind to soluble apaf-1, thereby activating caspases 9 and 3. In this respect, a reaction was obtained with an anti-apaf1 antibody in bull, boar, and rabbit CEP and an anti-caspase 9 antibody in bull CEP (Fig. 3, D and E). As the sizes of the antigens detected are different from their somatic counterparts, final identification must await sequence data. No reaction was detected with the anti-caspase 3 antibody (Fig. 3F), although this may be due to problems with cross-reactivity between species. Recent evidence indicates that, in nonproliferating terminally differentiated cells, there is interplay between the proteasomal and apoptotic pathways [71], the former representing the constitutive pathway for protein degradation and the latter an acute response to external stress.



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  		FIG. 3. Western blots of proteins in bull, boar, rabbit, and rat CEP probed with antibodies against (A) ubiquitin, (B) 20S proteasome, (C) survivin, (D) apaf-1, (E) caspase 9, (F) caspase 3, (G) control (i.e., second layer peroxidase-conjugated antibody only). Each lane contained 7.5 µg protein. Electrophoresis, Western blotting, and chemiluminescence detection procedures were performed as described previously [75]. Primary antibodies (rabbit polyclonal) were supplied by Abcam (Cambridge, UK) and had the following product numbers: ubiquitin, ab7780; 20S proteasome, ab3325; survivin, ab496; apaf1, ab2001; caspase 3, ab4051; caspase 9, ab2013. Horseradish peroxidase (HRP)-conjugated swine anti-rabbit IgG was obtained from DakoCytomation (Ely, UK). As shown in (G), there was artifactual binding of the second layer antibody to a 51-kDa protein in rabbit CEP. This appears in all the blots. Bull testicles were obtained within 2 h of slaughter and boar, rabbit, and rat tissues within 30 min of death. Luminal fluid was collected from the cauda epididymidis by retrograde injection of PBS (preceded by a small air bubble) via the vas deferens and sucking the contents that emanated from a small incision on the surface of the duct into capillary tubes. The tubes were centrifuged at 12 000 x g for 40 min and the supernatant plasma (CEP) removed and stored frozen at –20°C

Given the evidence for degradative pathways in CEP, are survival signals also present? So far, the only component reported is survivin (Fig. 3C), also known as BIRC-5, which is a member of the inhibitors of apoptosis (IAP) family of small molecular weight proteins that block apoptosis by binding to caspases. Survivin is expressed transiently during embryogenesis and is found in most tumors, but is rarely detectable in normal adult tissues [72]. Exceptions are thymus, spleen, and testis [73]. Nothing is known about epididymal tissue or mature spermatozoa. However, on Western blots of bull, boar, and rabbit CEP, a signal is present at the appropriate size for survivin (16.3 kDa), although final verification will require sequence data. The higher molecular antigens identified by the antibody in bull and rabbit CEP may represent survivin bound to other proteins. It remains to be demonstrated whether additional survival signals (e.g., Bcl-2) are secreted into CEP and if they are regulated by androgens.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 ARE THERE ANY CIRCUMSTANCES...
 HYPOTHESIS
 CONCLUSIONS
REFERENCES
 
The available evidence suggests that the necessary hydrolytic pathways for breakdown of sperm proteins and DNA are present in the CEP from normal animals. Because spermatozoa are not degraded under these circumstances to any appreciable extent (i.e., survival signals predominate), it supports the hypothesis that the changes that take place within the lumen of the duct following removal of androgen and regression of the epithelium activate death pathways that lead ultimately to dissolution of spermatozoa. In male animals with a seasonal reproductive cycle, this mechanism would come into operation as testosterone levels decline, thereby ensuring that the epididymis is cleansed of defective and dead spermatozoa in preparation for the next breeding period.

This hypothesis is open to experimental scrutiny. Should a signaling mechanism be present downstream of androgen action that controls the switch between survival and death signals, then it would be an obvious target for drug intervention that would tip the balance within the lumen of the cauda epididymidis to change it from an organ that normally preserves spermatozoa to one that actively destroys them.


   ACKNOWLEDGMENTS
 
I am grateful to colleagues Liz Howes and Peter James for helpful discussions and for preparation of the data shown in Figures 2 and 3. The views presented in this paper are entirely those of the author and are intended to stimulate alternative and novel lines of research in epididymal physiology to those currently in vogue.


   FOOTNOTES
 
1 This work was funded by the BBSRC. Back

2 Correspondence: Roy Jones, Laboratory of Molecular Signalling, The Babraham Institute, Cambridge CB2 4AT, UK. FAX: 44 0 1223 496022; roy.jones{at}bbsrc.ac.uk Back

Received: 23 April 2004.

First decision: 18 May 2004.

Accepted: 14 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 ARE THERE ANY CIRCUMSTANCES...
 HYPOTHESIS
 CONCLUSIONS
 REFERENCES
 

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