THE TESTIS/SPERMATID THIOREDOXIN SYSTEM IN MALE REPRODUCTIVE DISEASES

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There are two major pathological situations resulting as a consequence of failure or malfunctioning of male germ cells: infertility and testicular cancer. Sadly, human infertility is a fairly common condition which, by various estimates affects 15-20% of couples, with approximately equal contribution from both partners (18). Historically, failures related to germ cell formation have been studied more intensively in males than in females (68). One reason of this bias is the fact that the production of germ cells from the undifferentiated spermatogonia to the mature spermatozoa occurs during adulthood, whereas the production of the oocyte takes place during fetal time. In addition, the extracorporal position of the testis makes studies of gametogenesis in male more feasible than in female. Many environmental, behavioural and genetic factors affect male infertility and it has been estimated that the genetic factor accounts up to 60% of the causes underlying this phenotype, mostly due to autosomal-recessive genes (46). A reduction in sperm count and infertility has been found to be associated with an increased rate of chromosomal abnormalities (101). The rate of these abnormalities spans from 4.1% in men with oligozoospermia to 15.4% in azoospermic men. The causes can be diverse and might be related to failure of chromosome pairing and crossing-over in meiosis as well as chromosomal breakpoints in genes important for testicular development and function (68). Furthermore, saturation mutagenesis studies carried out in Drosophila indicate that the combined effect of many genes is likely to contribute in a much higher proportion to defects of spermatogenesis than effect of single genes (32).

Despite the fact that the Y chromosome has acquired a large number of testis-specific genes during recent evolution (89) none of the Sptrxs or Txl-2 genes are localized in this chromosome (Table I). Although we do not have evidence yet that any of the spermatid-specific thioredoxins are involved in male infertility phenotypes as a consequence of chromosomal abnormalities, their expression pattern supports a potential role in this pathology.

Among the pathologies affecting spermatozoa, dysplasia of the fibrous sheath (DFS) is an anomaly found in spermatozoa of severe asthenozoospermic patients characterized by a marked hypertrophy and hyperplasia of the fibrous sheath (76, 77). In addition to causing an abnormal configuration of FS, DFS affects various cytoskeletal components including axonemal microtubule doublets, ODF and the mitochondrial sheath (15). As mentioned above, Sptrx-1 and Sptrx-2 are two components of the sperm tail FS and Txl-2 is present in the transient spermatid manchette and in the tail axoneme. The localization of these thioredoxins in the spermatid tail makes them potential candidates to be involved in the development of DFS as it has been reported that a strong genetic component underlies this pathology (14). It will be very interesting to ascertain whether any mutation, polymorphism or any other genetic anomaly affecting any of the genes coding for the spermatid thioredoxins are correlated with DFS.

Primary ciliary dyskinesia (PCD), also known as immotile cilia syndrome (ICS) is a disorder affecting ciliary movement with an incidence of 1 in 20,000-30,000. Genetic studies demonstrate an extensive locus heterogeneity of this trait where the majority of affected families transmit PCD as an autosomal recessive disease (11). In PCD patients, cilia and sperm flagella demonstrate reduced motility due to diverse molecular pathologies often involving the dynein arms of axonemal microtubule doublets, resulting in chronic respiratory problems, dextrocardia and situs inversus, hydrocephalus and male infertility, (11). Indeed, DFS has also been considered as a variant of PCD as the absence of dynein arms in axonemes is a common symptom (16). The FS location of Sptrx-1 and Sptrx-2 and its potential involvement in DFS makes it possible, although less likely, that they participate in PCD. However, Txl-2 is a serious candidate gene for PCD due to its axonemal localization and its microtubule-binding activity (82).

Sperm-flagellar pathology is often associated with the retention of redundant cytoplasm that would otherwise be rejected as residual body and cytoplasmic droplet during final stages of spermiogenesis. This is well illustrated by the association of Sptrx3 with the redundant cytoplasm and nuclear vacuoles in sperm from teratospermic infertility patients (Fig. 5; Sutovsky and Miranda-Vizuete, unpublished results).

Another anomaly affecting male reproductive function is the development of autoimmune antibodies to spermatozoa (antisperm antibodies). Being associated with the impaired sperm function at various stages of reproductive process, autoimmune disease is another important cause of male infertility (17). There are two types of antisperm antibodies: those induced as a consequence of obstruction of the male reproductive tract by disease, trauma or surgical procedures such as vasectomy (also denominated sperm autoantibodies) (26, 27) and those produced by the female partner, interfering with the normal transit through the female reproductive tract and sperm-oocyte recognition (17). The production of male sperm autoantibodies is assumed to be a consequence of stimulation of the immune response when the spermatozoa or their components are no longer sequestered behind the blood-testis and blood-epididymal barriers. The male duct system suffers frequent ruptures after obstruction forming spermatic granulomas in which sperm come in contact with macrophages, lymphocytes and other immune cells (27). Not much is known about the causes by which female body induces the production of antisperm antibodies. Most often, it is assumed that the immune response is mounted as a consequence of previous exposure to sperm antigens when the female mechanisms that normally tolerate the haploid “foreign” spermatozoa do not function properly or become hypersensitive (88). Other explanations have been considered such as molecular mimicry by which epitopes from invading pathogens bear similarity to those of spermatozoa thereby leading to antibody cross-reaction (17).

As sperm tail outer dense fibers have been reported to be the dominant postobstructive autoantigens (27), we wondered whether any of the spermatid-specific thioredoxins (which are located in the FS of the spermatid tail externally surrounding ODF) can also be categorized in this group. This approach has resulted in identifying Sptrx-2 as a novel sperm autoantigen, while antibodies recognizing Sptrx-1 or Txl-2 were not detected in the post-vasectomy rat sera (Miranda-Vizuete et al., submitted for publication). This result indicates that not only ODF but also some FS proteins are able to elicit sperm autoantigens and therefore should be taken into consideration when screening for novel component of the postobstructive autoimmune response.

The other major pathology affecting the male reproductive system are testicular germ cell tumors (TGCTs), which are not a direct cause of male infertility but their treatment and management can impair or eradicate spermatogenesis. Although germ cell tumors are rare in the general male population as a whole, accounting for less than 1% of all cancers, they are the most common malignancy in young adult Caucasian males. They are mainly found during the 3^rd to the 4^th decade of life with an incidence of 6 to 11 per 100 000 and there is a continuous increasing trend (10, 50). TGCTs are classified into three groups by epidemiological, clinical and histological studies: a) teratomas and yolk sac tumors which always manifest before puberty, b) seminomas and nonseminomas that appear after puberty and c) spermatocytic seminomas which usually appear in elderly men (51). Seminomas and nonseminomas account for the vast majority of the TGCTs while yolk sac tumors, teratomas and spermatocytic seminomas are rare. It is now generally agreed that both seminomas and nonseminomas originate from carcinoma in situ (CIS) while the origins for the other two, rare types of testicular tumors are still not clear but definitively not CIS (78). CIS cells are localized within the seminiferous tubules between the basal membrane and the Sertoli cell layer and resemble early primordial germ cells. It is assumed that the initiating event leading to the development of CIS originates during intrauterine development (39, 78). TGCTs are uniquely sensitive to cisplatin-based chemotherapy with more of than 90% of newly diagnosed cases cured (75). This property makes the TGCTs an ideal system to study cell death pathways and their relevance to the treatment of other types of cancer (75). The major cytotoxic effect of cisplatin is generally attributed to the formation of DNA-platinum adducts which cause cell cycle arrest and trigger apoptosis (5). Cisplatin is an efficient inhibitor of both Trx-1 and TrxR1 as well as glutaredoxins, and the cytosolic thioredoxin and glutaredoxin systems have been implicated in the cellular pathways leading to cisplatin detoxification (5, 85, 86). Thus, increased expression and activity of the thioredoxin system has been correlated with resistance against cisplatin-induced cytotoxicity by tumor cells (84, 108). Moreover, some studies have identified chromosomal amplifications in resistant TGCTs affecting Trx-1 and TrxR1 locus (75, 92). In this context, the presence of four testis/spermatid-specific thioredoxins raises the possibility that abnormal levels of one or several of these novel thioredoxins might be involved in the 10% of the TGCTs that are resistant to cisplatin treatment. To examine this hypothesis, we have initiated a study aiming to determine the mRNA and protein levels of the four spermatid-specific thioredoxins in all types of testicular tumors, which is expected to shed more light into the biochemical mechanisms that result in drug resistance.