Molecular Human Reproduction, Vol. 8, No. 8, 695-701,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Review article |
The dog as a model to study human epididymal function at a molecular level
IHF, Universität Hamburg, Grandweg 64, D-22529 Hamburg, Germany. E-mail: kirchhoff{at}ihf.de
Abstract
Approaches to study human epididymal functions are limited. Therefore, suitable animal models are highly desirable, yet difficult to find among the few species studied on a molecular level to date. This review summarizes our progress in the development of the canine epididymis as an alternative model. Dogs are biomedically a key species because they are subject to many of the same diseases as humans and already serve as a model for a number of human pathologies, including genetic diseases. It is thus consistent that an appraisal of epididymal specific gene expression has been started in the dog, including the molecular cloning and characterization of canine epididymal proteins. These proteins, in addition to a high overall sequence similarity to the human, show a similar tissue distribution, relative abundance and spatial pattern within the epididymis. Moreover, the dog epididymis offers an excellent source for cell culture studies, and immortalization of the canine epididymal duct epithelium has been achieved, encouraging regulatory studies of epididymal gene expression in vitro. Thus, the dog already fulfils many of the criteria of a good model of the human epididymis on a molecular level. Further progress may be expected with the advance of the canine genome project. If there is a genetic basis for male infertility, then the dog provides the advantage of the exploitation of a species that combines a maximum of genetic variation within a species with the capacity to minimize that variation within a defined pedigree.
animal model/dog/epididymis/gene expression
Introduction
For in-vivo fertilizing capacity, mammalian sperm require a post-testicular maturation process, including their exposure to the specific microenvironment provided by the epididymis. There is ample circumstantial evidence that epididymal proteins are involved in this process (Kirchhoff, 1999
; Dacheux and Dacheux, 2002). However, our understanding of how the individual proteins implement the acquisition of sperm fertilizing ability is still fragmentary. In order to better appreciate the role of the epididymis at a molecular level, a careful analysis of its specific pattern of gene expression is helpful. As in many other fields of biomedical research, rodents, specifically the mouse, have long been the preferred models for studying epididymal gene expression due to the feasibility of animal experiments including targeted gene disruption. However, because of profound differences in physiology, induced targeted mutations in the mouse may not always be of direct relevance to human disease. Considering also the enduring disputes on phylogenetic relationships among the orders of placental mammals (Springer and de Jong, 2001), it is highly desirable to develop alternative animal models.
Since the 17th century, dogs have been used as models in physiological and metabolic studies. Because of obvious parameters such as lifespan and size they are an important intermediate species between the human and the mouse. In dogs, the physiology, disease presentation and clinical response seem to be closer to the human (Ostrander and Giniger, 1997). The list of how the dog is presently being used to study human health and disease is long, including fields as different as ageing, pharmacology, toxicology, neurology, cancer and genetic diseases. Often, a dog disease represents a naturally occurring model for a similar disease in the human. At present, >370 inherited diseases of dogs are being catalogued. Some of these diseases are found only in dogs and humans, such as X-linked retinal degeneration. Considering that the canine genome is organized in a similar way to that of humans, many dog diseases closely resemble human genetic disorders (Ostrander and Giniger, 1997). Fifty-eight percent of dog genetic diseases, such as Duchenne-type muscular dystrophy, are true homologues of human diseases caused by mutations in the same gene. A linkage map of the canine genome has recently been published (Breen et al., 2001) and in the future will allow the exploitation of the dog as a powerful system to map complex genetic traits, possibly including male subfertility and infertility. These traits are likely to have related causes in humans and dogs, and localizing the related genes will be much easier in dogs than in humans because of large purpose-bred pedigrees and selective inbreeding.
Canine epididymides can be obtained from veterinary practices where dogs are being castrated for behavioural disturbances, thus offering a convenient and acceptable tissue source for various molecular and cellular studies. An appraisal of canine epididymal gene expression was started (Ivell et al., 1998; review), initially with the aim of achieving a better understanding of species differences and similarities in male reproductive genes. From these studies, the dog epididymis turned out to represent a useful model with high relevance to humans, especially at the level of tissue-specific gene expression. In the future this should help us to better understand the molecular basis of human reproduction, including male idiopathic subfertility and infertility.
Gene expression in the canine epididymis—basic molecular biology
Molecular cloning of canine epididymal proteins by homology to the human
Gene expression in the canine epididymis was initially studied by looking for genes homologous to those known in the human (Kirchhoff et al., 1990, 1991, 1993; Ellerbrock et al., 1994; Osterhoff et al., 1997) (Table I). Similar to their human counterparts (Krull et al., 1993), the cloned canine epididymal (Ce) mRNAs were of epithelial origin and showed characteristic spatial patterns along the epididymal duct (Pera et al., 1994). Ce1, the canine homologue of human epididymal HE1, predicted a highly abundant, conserved secretory protein (Ellerbrock et al., 1994; Ivell et al., 1998). The mRNA was found in large amounts in the epididymal duct epithelium (Pera et al., 1994; Figure 1), while the protein was largely found in the duct lumen. Homologous proteins have been linked to epididymal luminal cholesterol transfer in various mammalian species (Okamura et al., 1999). Recently, HE1 has been identified as the second gene of the Niemann-Pick type C disease (NPC-2) involved in cholesterol egress from lysosomes (Naureckiene et al., 2000). Considering the very high Ce1 expression levels (
5% of Ce mRNA) (Ellerbrock et al., 1994), cholesterol transfer or exchange appears to be a major function of the canine epididymis.
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Ce4 encodes a less abundant and less well-conserved protein containing two tandem-arranged whey-acidic-protein (WAP) domains (Ellerbrock et al., 1994
). Similar to Ce1, the Ce4 mRNA was found at similar levels in most regions of the canine epididymis (Pera et al., 1994; Figure 1
). The predicted peptide sequence showed significant motif similarity to the secretory leukocyte proteinase inhibitor (SLPI/ALP/HUSI-1) suggesting that it might represent a proteinase inhibitor of the male genital tract. Proteases have important functions in epididymal sperm maturation (Metayer et al., 2002). Their regulation by specific inhibitors, including Ce4, might control their activity in vivo. HE4 shows genetic linkage to the SLPI gene on human chromosome 20. Very close to this chromosomal region lie several other genes encoding proteinase inhibitors and major secretory proteins of the human epididymis, suggesting that this region may be important for male reproduction (Richardson et al., 2001). It will be interesting to see whether synteny of homologous genes is revealed by linkage mapping of the canine genome (Ostrander and Giniger, 1997; Langston et al., 1999).
The Ce5 cDNA encodes the counterpart of the glycosylphosphatidylinositol (GPI)-anchored HE5/CD52 major sperm membrane antigen (Kirchhoff, 1996; Schröter et al., 1999). HE5 was the first well-defined sperm membrane glycopeptide implicated in human antibody-mediated infertility (Diekman et al., 2000; review) and is a major component of the glycocalyx of functionally mature sperm (Kirchhoff and Schröter, 2001). In contrast to the common opinion that epididymal proteins are only loosely associated with the sperm surface, this membrane antigen, despite its post-testicular origin, becomes an integral part of the sperm plasma membrane by means of its GPI anchor (Kirchhoff and Hale, 1996; Yeung et al., 1997). Like its human homologue, Ce5/CD52 showed a regionalized expression with maximum levels in the distal corpus and cauda epididymidis (Pera et al., 1994; Figure 1). From the conserved parts of its cDNA sequence (Ellerbrock et al., 1994), it may be inferred that, irrespective of its completely divergent mature peptide sequence, Ce5 is also GPI-anchored.
Ce6 represents the homologue of the human epididymis-specific heptahelical receptor HE6 (Osterhoff et al., 1997) which is a key component in a signal transduction pathway controlling epididymal function and male fertility. The canine cDNA sequence has only been partially characterized (unpublished results). From this partial sequence, however, the seven transmembrane-coding region of the canine receptor appears to be nearly identical to that of the human (>95% amino acid identity), suggesting a very high overall sequence conservation of this orphan receptor.
Cloning of novel cDNAs of the canine epididymis
A differential screening procedure was later applied to the dog epididymis with the aim of cloning novel tissue-specific cDNAs, which may have escaped previous approaches in other species. Out of the products identified, six cDNAs were studied in greater detail and were derived from abundant, i.e. Northern-detectable, mRNAs, namely Ce7-Ce12 (Table I). With the exception of the epididymal secretory glutathione peroxidase GPX5, which according to our nomenclature is referred to as Ce7 in the dog (Beiglböck et al., 1998; Table I), they indeed represented novel gene products not previously found in the databases. GPX5 is part of the hydrogen peroxide scavenging system found within the epididymides of most mammalian species studied. However, the encoding mRNA was found in the proximal parts of the canine epididymis, which is different from rodents where it is restricted to the caput region (Beiglböck et al., 1998; Figures 1 and 2).
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The Ce8–Ce11 cDNAs predicted novel epididymal proteins whose functions still remain speculative. The spacing pattern of cysteines in Ce8 suggests that it belongs to the Ly-6-domain superfamily of proteins, which includes the sperm-specific antigen SP-10 (Freemerman et al., 1995
). The predicted Ce9 peptide (not related to the `CE9' membrane protein of rat sperm) (Petruszak et al., 1991) is highly hydrophobic and may comprise four membrane-spanning domains (Gebhardt et al., 1999) similar to the 19.5 protein of the mouse (MacLeod et al., 1990). Different from the other Ce mRNAs, Ce8 and Ce9 display highly restricted and congruent expression patterns in that both mRNAs were exclusively found in the utmost proximal part of the canine epididymis (Figures 1 and 2). The Ce10 cDNA sequence predicts a small secretory protein distantly related to the second WAP domain of Ce4; however, unlike Ce4, it contains only one such domain (Gebhardt et al., 1999). The mRNA was expressed only in the distal parts of the canine epididymis. The Ce11 cDNA sequence again predicts a large membrane protein, which is similar to the 12-transmembrane co-transporters and is expressed in all parts of the epididymal duct (unpublished results).
Ce12 is described in greater detail here, since it was used to detect a highly homologous human counterpart (Saalmann et al., 2001). Ce12a and b cDNAs predict novel epididymis-specific fibronectin type II (Fn2) proteins, the different isoforms originating from alternative mRNA splicing and/or usage of two alternative promoters. Maximum levels of all mRNA variants are found in the corpus region (Figure 1). The encoded proteins represented the first known examples with four tandemly-arranged Fn2 domains, closely similar, but not homologous, to the ungulate seminal plasma proteins (Salois et al., 1999; 50% sequence identity) which have been implicated in sperm capacitation (Töpfer-Petersen et al., 1995; Therien et al., 1998). Antibodies were raised against the predicted proteins and employed in Western blot analysis and immunohistochemistry (Figure 3). Multiple peptide isoforms were detected in protein extracts of whole epididymides, in epididymal luminal fluid as well as on epididymal sperm, confirming that Ce12 is a secreted protein which binds to sperm. This accords with the majority of other mammalian Fn2 module proteins, 90% of which are secretory.
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Good applications and exceptions
Similarity of epididymal protein expression in dogs and men
During our search for an animal model with relevance to the human, the canine counterparts of HE1, HE4 and HE5/CD52 were identified by cross-hybridization in a Northern blot employing the human cDNAs as heterologous probes (Kirchhoff et al., 1990, 1991, 1993). This suggested that the corresponding canine homologues were both sufficiently similar and abundant. Consistent with this observation, their cDNAs were readily obtained by standard homology screening of a canine epididymal cDNA library (Ellerbrock et al., 1994). More detailed studies revealed that their tissue distribution, as well as their relative abundance and spatial patterns within the epididymides, were very similar in both species (Krull et al., 1993; Pera et al., 1994; Ivell et al., 1998). To emphasise their homology with the human, the canine gene products were named Ce1, Ce4 and Ce5/CD52 (Table I). Different from homologues of other species, the Ce mRNAs were similar to the human even in their 3' untranslated sequences (Kirchhoff et al., 1990, 1993, 1996; Uhlenbruck et al., 1993), suggesting that post-transcriptional regulatory mechanisms might also be comparable in this species. The corresponding rodent sequences, in contrast, had diverged to a much greater extent.
Reversing the cross-hybridization procedure and employing in turn the Ce cDNAs as probes in the human, out of the novel Ce7–Ce12 cDNAs (Table I), only the Ce12 probe readily detected an abundant human epididymal counterpart, named HE12 (Saalmann et al., 2001; see above). Homologous counterparts have also been found in other mammals, although rodent homologues have remained elusive. The HE12 mRNA was epididymis-specific, as were the Ce12 variants, and showed a similar expression pattern with maximum expression levels in the human corpus. HE12 protein is also secreted and was detected in human epididymal luminal fluid as well as on ejaculated sperm, and multiple secretory protein isoforms were detected for both HE12 and Ce12, suggesting similar post-translational modifications.
Differences between human and canine patterns of epididymal gene expression
No functional human homologues have as yet been detected for Ce7/GPX5, Ce8 and Ce9 (compare Table I). However, failure to detect human counterparts to caput-expressed epididymal proteins does not seem to represent a specific constraint of the dog model. Rather, the highly abundant proximal gene products cloned from rodents and non-human primates have also failed to identify abundant human counterparts (Hall et al., 1998; Lareyre et al., 1998; Perry et al., 1999). The human caput has a distinct epithelial morphology (Holstein, 1969; Yeung et al., 1997), hence its epididymal duct epithelium may also be functionally distinct and express different genes. Thus, it seems to be the expression pattern of the human caput, which is different from that of other mammals.
When employed in database screenings, the Ce7/GPX5, Ce8 and Ce9 sequences, although not identifying (functional) human mRNAs by homology screenings of epididymal cDNA libraries, still identified closely related (partial) express sequence-tagged clones and/or human genomic sequences (compare Table I). This apparent discrepancy may be explained by the observation that the human genome appears to contain a relatively high proportion of pseudogenes that have active homologues in other mammalian species (Hacia, 2001; review). Differences in gene regulation and splicing are yet other mechanisms that could profoundly affect epididymal gene expression, and gene functions can be lost or gained by subtle changes such as point mutations in non-coding sequences, e.g. promoters (Huby et al., 2001). These mechanisms may apply to certain epididymal gene products due to a reduced selective pressure in the human. Additional differences in HE and Ce gene expression may result from the fact that the dog has no seminal vesicles. The induction of seminal vesicle-like glandular structures and secretory proteins from the canine epididymal duct epithelium was achieved by heterotypic mesenchymal induction (Chan and Wong, 1996), indicating a remarkable degree of flexibility in the epithelial differentiation of the male genital tract. However, it is unknown whether the more distal parts of the canine epididymis substitute for secretions which in the human derive from seminal vesicles.
Factors modulating Ce gene expression
Hormonal status and positional information are major factors regulating epididymal gene expression (Kirchhoff, 1999; review). It can be inferred that this is also true in the dog. The presence of various steroid receptors in the canine epididymis has been known for some time (Younes et al., 1979) and a few publications describe dramatic effects of hormonal treatment on the morphology and histology of the epididymis in intact animals (Connell and Donjacour, 1985). A recent paper describes the differential expression of estrogen receptor 
and ß proteins in the epididymis of the adult male dog (Nie et al., 2002). However, hormonal effects on the Ce gene products cannot easily be studied because animal experimentation in the dog is not unproblematic for ethical and practical reasons. Our knowledge of in-vivo regulation or modulation of Ce gene expression therefore comes solely from naturally occurring pathological conditions (Pera et al., 1996; Beiglböck et al., 1998; Gebhardt et al., 1999) and from studies of animals of different ages (Gebhardt et al., 1999).
A pathological condition with high relevance to Ce expression is the failure of testicular descent (cryptorchism). This defect appears to have a genetic component and is propagated at a relatively high incidence as a consequence of common breeding practices in certain dog breeds. In a proportion of afflicted animals, the hemi-cryptorchids, only one testis and epididymis fail to descend into the scrotum, thus after castration providing test tissue and an endogenous control from the same animal. In a study involving hemi-cryptorchid dogs without testicular cancer, the majority of Ce mRNAs with spatial restriction in the epididymis, namely Ce7/cGPX5, Ce8, Ce9 and Ce10, were markedly reduced in the abdominal organs (Figure 1). Although the hormonal status of these dogs was not recorded, this decline most probably occurred in response to temperature elevation and/or long-term withdrawal of various testicular factors, other than steroid hormones. Regionalization within the epididymis is characteristic of most Ce mRNAs (Figures 1 and 2). Interestingly, the effects of the abdominal position of testis and epididymis seem to be superimposed by the effects of regionalization, i.e. a reduction of Ce mRNA levels in the abdominal epididymides was most obvious in the caput region (Figure 1).
Regarding the effects of ageing on Ce mRNA levels, only the caput-expressed Ce7/cGPX5 and Ce8 mRNAs were significantly reduced in dogs of increasing age, while most other mRNAs were not affected, and Ce4 levels even appeared to increase with age (Gebhardt et al., 1999). Although these results were based on a relatively small number of individuals, they still suggest that relative mRNA levels reflect the age of an animal, and that age-related effects vary depending upon the epididymal region studied (Gebhardt et al., 1999). Since the human epididymides employed in previous molecular studies were derived from elderly men, these observations could also be of relevance to results obtained in the human. Many studies have revealed an age-dependent decrease in free testosterone levels in the human male. Unfortunately, in dogs there are conflicting results concerning the effect of ageing on hormone levels. Both a decrease of LH and testosterone levels with age (Günzel-Apel et al., 1990) and no age-related changes except for decreasing estradiol levels (Peters et al., 2001) have been reported. Thus, the usefulness of the ageing dog as a model to study the effects of ageing on human epididymal sperm maturation and storage still remains to be established.
Cell culture systems to study regulation of epididymal gene expression
Representing a relatively large organ with a high proportion of epithelial cells, the dog epididymis offers an excellent source for primary cell culture studies. The cultured cells grow by division as well as by migration, and in our hands fibroblast overgrowth does not represent a problem, even in the presence of serum in the culture medium (Pera et al., 1996). The cultured cells show important characteristics of the native epididymal duct epithelium, namely an epitheloid morphology, expression of an epithelial cytoskeleton and nuclear androgen receptor, and can be maintained for sufficient time periods for short- to medium-term experiments. Most interestingly, they allowed us to demonstrate for the first time a direct effect of temperature on cells of epididymal duct origin, independent of temperature effects on the testis (Pera et al., 1996). Exposure to a culture temperature of 37°C (abdominal temperature) compared with 33°C (scrotal temperature) had a fast and irreversibly suppressive effect on the levels of the Ce5/CD52 mRNA encoding the major `maturation-associated' sperm membrane antigen (Table I). This temperature effect on Ce5 was a direct and specific one, not involving other epididymal gene products. Treatment of the cells with transcriptional and translational inhibitors suggested that the steady-state levels of Ce5/CD52 mRNA were controlled post-transcriptionally (Pera et al., 1996).
A major obstacle of primary cultures, however, is their relatively short duration and finite cell number due to the limited proliferative potential of the cultured cells. We therefore generated permanent cell populations of the differentiated, adult canine epididymal epithelium. Immortalization was accomplished by retroviral infection with the SV40 large T antigen, and the cell lines obtained have been named IMCE for immortalized canine epididymis (Telgmann et al., 2001). The IMCE cell lines analysed reflected at least all functions of the primary cell cultures. However, compared with the original tissues, their levels of Ce1 mRNA were reduced while Ce4 mRNA levels had increased. In addition to the nuclear androgen receptor, the polyoma enhancer activator (PEA3) and as the estrogen receptors ER and ERß were detected at the transcript level. The majority of the IMCE cells were ER-positive but ERß-negative, while one ER-negative IMCE population was positive for ERß (Telgmann et al., 2001). Thus they may represent useful permanent tools for studying gene regulation by in-vitro promoter studies and for other types of experiments, including hormonal, drug and toxicity effects. Epithelial cells of the adult canine epididymis are ER-negative while those of the efferent ducts are strongly positive (Nie et al., 2002). It will be interesting to see whether one of the ER-positive IMCEs could be of efferent duct origin.
Conclusions
The dog fulfils many of the criteria of a good animal model to study epididymal gene expression. Further development of such models will ultimately permit any human epididymal expressed gene of interest to be studied in the animal species most suitable for its analysis. Of all the species we have studied, the dog has by far the highest number of closely related counterparts to the cloned HE proteins. This is by no means self-evident, since it has long been observed that there is poor conservation among mammals of some of the most abundant epididymal proteins (Brooks et al., 1986a,b; Kirchhoff et al., 1991; Hayashi et al., 1996; Kirchhoff and Hale, 1996). Indeed, a high level of sequence divergence appears to be characteristic of male reproductive proteins (Rooney and Zhang, 1999; Swanson et al., 2001), sometimes even between closely related species (Sutton and Wilkinson, 1997; Wyckoff et al., 2000). Positive Darwinian selection may be the driving force behind the rapid evolution of these proteins (Wyckoff et al., 2000).
The Ce cDNAs and cell culture systems described here represent valuable tools to study epididymal functions on a molecular level. Initial studies involving epididymides from a wide variety of dog breeds and mongrels suggested that tissues can probably be taken from any breed with expectation of comparable results at the molecular level (Ellerbrock et al., 1994). More recent studies involving the novel Ce products with their more restricted spatial patterns (Gebhardt et al., 1999) showed a much higher degree of variation in their mRNA levels. Indeed, if there is an epididymal contribution to male fertility variation and to the more specific cases of idiopathic male infertility, then these variations in Ce mRNA levels may represent the raw material to study. The diversity among the >300 modern dog breeds will provide an important reservoir of genetic differences which can be exploited in relation to the natural variation of epididymal functions as well as to its toxicology and the effects of drugs. In addition, besides a contribution to human biomedical research, the results summarized here may also provide a basis for future contraceptives in the dog, and thus also be of benefit to dog owners and breeders (not to mention dogs).
Acknowledgements
The author is grateful to her present and past co-workers for their efforts in developing the dog model, as well as to the local veterinarians for their continuous support. Professor Dr Richard Ivell, IHF, Hamburg and Professor Dr Edda Töpfer-Petersen, Tierärztliche Hochschule Hannover, contributed by many stimulating discussions. Professor Dr Freimut Leidenberger, IHF, provided excellent working facilities. The work presented was supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG contract no. Ki 317/5).
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Submitted on February 4, 2002; accepted on May 3, 2002.
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