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BOR - Papers in Press, published online ahead of print March 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040360
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BIOLOGY OF REPRODUCTION 73, 201–211 (2005)
DOI: 10.1095/biolreprod.105.040360
© 2005 by the Society for the Study of Reproduction, Inc.

Selenoprotein P Is Required for Mouse Sperm Development1

Gary E. Olson 2 3, Virginia P. Winfrey 3, Subir K. NagDas 3, Kristina E. Hill 4, and Raymond F. Burk 4

Department of Cell and Developmental Biology3 Division of Gastroenterology,4 Vanderbilt University, Nashville, Tennessee 37232-2175


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selenoprotein P (SEPP1), an extracellular glycoprotein of unknown function, is a unique member of the selenoprotein family that, depending on species, contains 10–17 selenocysteines in its primary structure; in contrast, all other family members contain a single selenocysteine residue. The SEPP1-null (Sepp1–/–) male but not the female mice are infertile, but the cellular basis of this male phenotype has not been defined. In this study, we demonstrate that mature spermatozoa of Sepp1–/– males display a specific set of flagellar structural defects that develop temporally during spermiogenesis and after testicular maturation in the epididymis. The flagellar defects include a development of a truncated mitochondrial sheath, an extrusion of a specific set of axonemal microtubules and outer dense fibers from the principal piece, and ultimately a hairpin-like bend formation at the midpiece-principal piece junction. The sperm defects found in Sepp1–/– males appear to be the same as those observed in wild-type (Sepp1+/+) males fed a low selenium diet. Supplementation of dietary selenium levels for Sepp1–/– males neither reverses the development of sperm defects nor restores fertility. These data demonstrate that SEPP1 is required for development of functional spermatozoa and indicate that it is an essential component of the selenium delivery pathway for developing germ cells.

epididymis, gamete biology, sperm, sperm maturation, spermatid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selenium is a dietary micronutrient required by mammalian tissues and is essential for the maintenance of normal spermatogenesis and male fertility. Early studies demonstrated that selenium is incorporated into mouse and rat spermatogenic cells [1, 2] and that, in selenium-replete adult rats, the testes and spermatozoa contained higher selenium concentrations than other major organs [3, 4]. It was also demonstrated that the spermatogenic pathway claimed a much greater fraction of available selenium in selenium-deficient rats than in controls [2] and that testes maintained near-normal selenium levels, whereas levels in other organs were falling precipitously [5]. Thus, the testis has a mechanism by which it competes effectively for selenium within the animal to support spermatogenesis. Even so, selenium deficiency results in production of abnormal spermatozoa [6–9], and prolonged deficiency results in atrophy of the seminiferous epithelium [10].

Selenium exerts its biological function as the amino acid selenocysteine that is incorporated into the primary structure of members of the selenoprotein family [11]. More than 20 selenoproteins have been identified, and they include key enzymes for oxidant defense, such as the glutathione peroxidases and the thioredoxin reductases [12, 13]. Selenoproteins are usually enzymes that contain a single selenocysteine as a constituent of the active site. SEPP1 is a unique family member that is an extracellular glycoprotein and, depending on the species, contains 10–17 selenocysteine residues in its primary structure [14–16]. Although its specific function(s) remains to be identified, SEPP1 has been proposed both to perform an antioxidant role and to transport selenium throughout the body [17]. There are two proteins in blood plasma that contain selenocysteine, SEPP1 and the extracellular glutathione peroxidase (GPX3). SEPP1 accounts for most of the plasma selenium content [14], and its plasma half-life of approximately 4 h is consistent with either its catabolism or distribution to other tissues [18]. The liver synthesizes most of the plasma SEPP1 [19]; however, local production of the protein has also been noted in several other organs [20, 21]. Four isoforms of SEPP1 have been described that are apparently encoded by the same mRNA, but why four isoforms exist is not known [22, 23]. A study in rats that compared the tissue distribution of 75Se-labeled SEPP1 and GPX3 after injection into the femoral vein demonstrated a preferential uptake of SEPP1 by the testis [18], but neither the mechanism of its uptake nor the fate of its selenium has been studied. Likewise, SEPP1 uptake by the epididymis has not been examined. Recently, animals lacking the SEPP1 gene (Sepp1) have been produced independently by two groups. Both studies noted low selenium levels in several organs of Sepp1–/– animals [24, 25]; however, one also reported a profound loss of fertility in homozygous males [25]. The mechanisms underlying the SEPP1-dependent male infertility phenotype have not been identified.

Both the testis and epididymis require exogenously supplied selenium to synthesize a variety of selenoproteins, whose precise roles during spermiogenesis and after testicular sperm maturation are not clearly defined. The testis expresses key enzymes required for synthesis of selenoproteins, including selenocysteine ß-lyase [26], which is necessary when selenocysteine, presumably derived from selenoproteins that undergo proteolytic degradation, is the form of selenium supplied. The predominant testicular selenoprotein, phospholipid hydroperoxide glutathione peroxidase (GPX4, also termed PHGPx), is expressed predominantly by germ cells. Early studies demonstrated that selenium is concentrated in the sperm midpiece [2], where it is incorporated into a 20-kDa structural protein of the disulfide bond-stabilized mitochondrial capsule [27]; later, proteomic analyses identified this polypeptide of mature spermatozoa as an enzymatically inactive GPX4 [28]. Some of the GPX4 is also reported to localize to the spermatid nucleus, but its functional role too remains unclear [29– 32]. GPX4 appears critical to sperm function, because reduced GPX4 levels in human spermatozoa have been linked to male infertility [33–35]. Other sperm selenoproteins are less well characterized. Since spermatozoa contain several members of the thioredoxin family [36], they are likely to contain thioredoxin reductases(s), the selenoenzyme required for thioredoxin function. Thioredoxin reductase expression has been demonstrated in the testis [37] but its cell-specific distribution has not been determined. SEPP1 mRNA is also expressed in the testis, specifically by Leydig cells [38], and in vitro Leydig cell SEPP1 expression is regulated by cAMP [39]. Whether Leydig cells produce and/or secrete specific isoforms of SEPP1 has not been demonstrated. The selenoprotein complement of the epididymis has not been studied in detail. Principal cells express the selenoprotein GPX3 [40, 41], and although its role in oxidant defense may facilitate sperm survival, this possibility has not been demonstrated experimentally. Whether specific cell types or regions of the epididymis express other selenoproteins, including SEPP1, is an unresolved question.

A physiological function for SEPP1 in the testis or epididymis has not been established. However, since Sepp1–/– male mice are infertile [25], this study was undertaken to identify potential sperm abnormalities in null animals. We report that spermatozoa of SEPP1-null animals progressively develop a set of flagellar defects that resemble those seen in selenium-deficient wild-type mice. Moreover, neither the sperm defects nor infertility of Sepp1–/– males are reversed by prolonged dietary supplementation with selenium in the form of sodium selenite. We propose that SEPP1 plays an essential role in male fertility by providing a selenium source to the seminiferous tubule that is used by spermatogenic cells to synthesize their selenoprotein complement.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Care and use of animals conformed to National Institutes of Health guidelines for humane animal care and use in research, and all animal protocols were approved by the Vanderbilt Institutional Animal Care and Use Committee. Mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) approved facility on a 12L:12D cycle, and food and water were provided ad libitum. Sepp1–/– mice were produced and genotyped by polymerase chain reaction as described previously [25]. Sepp1–/– and Sepp1+/+ offspring of heterozygous parents fed Purina rodent chow were fed a Torula yeast-based diet supplemented with 1.0 mg/kg of selenium as sodium selenite [42] beginning at weaning. Males were examined between the ages of 2 and 12 mo.

To produce selenium-deficient mice, weanling C57B1/6 males (The Jackson Laboratory, Bar Harbor, ME) were fed ad libitum a Torula yeast-based selenium-deficient diet [42], and control mice were fed the same diet supplemented with 0.25 mg/kg of selenium as sodium selenite. At least 3 mice were examined for each of the selected time points between 0 and 15 mo.

Mice were anesthetized with isoflurane and exsanguinated by withdrawal of blood from the inferior vena cava. Blood was treated with disodium EDTA (1 mg/ml) to prevent coagulation, and after centrifugation the plasma was removed. Livers were removed and homogenized with 9 parts (w/w) of 0.1 M potassium phosphate, pH 7.0, and then centrifuged at 16 000 ;ts g and the supernatant was saved. The activity of two selenoenzymes was monitored to verify a selenium-deficient status [43]; glutathione peroxidase activity was measured in plasma (GPX3) and in the liver homogenate (GPX1) using 0.25 mM hydrogen peroxide as substrate.

Photomicroscopy

Testes and the caput and cauda regions of the epididymis were placed in Tyrode solution or Dulbecco PBS (145 mM NaCl, 4 mM KCl, 10 mM sodium phosphate, pH 7.4) at 37°C and gently minced with a razor blade. Spermatozoa were examined for motility and fixed by addition of 2 volumes of 4% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4).

Testes were also fixed in Bouin solution and embedded in paraffin, and sections were stained with hematoxylin-eosin. Photomicrographs were obtained using a Zeiss Axiophot and a Spot 2 digital camera (Diagnostic Instruments Inc., Sterling Heights, MI).

Electron Microscopy

Testes and epididymides were fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, postfixed in 1% OsO4 in cacodylate buffer, dehydrated through an ethanol series, equilibrated in propylene oxide, and embedded in Embed 812 (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were stained with uranyl acetate and lead citrate.

Fertility Analysis of Sepp1–/– Males

When the mice reached an age of 3–4 mo, they were exposed to C57Bl/6 female mice for periods of 4 days. The female mice were examined for evidence of pregnancy beginning at Day 14 after the initial exposure to a male. If there was no evidence of a pregnancy by Day 21 after the last exposure to a male, then the female was exposed to a different male for an additional 4-day period. Each male was mated with at least five different females, and the number of pregnancies and progeny were recorded.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chronological Development of Flagellar Defects in Spermatozoa of Sepp1–/– Mice

Light and electron microscopic comparisons of testicular and epididymal spermatozoa of wild-type and Sepp1–/– mice revealed a progressive development of severe flagellar defects in the null animals. Null animals between 2 and 12 mo of age exhibited identical sperm defects. Phase-contrast images of testicular spermatozoa from SEPP1-null mice revealed that they possessed a fully formed and extended flagellum; however, unlike spermatozoa from control mice, they frequently displayed a short, variable-length narrowing of the posterior midpiece; this narrowing appeared to reflect an absence of the mitochondrial sheath, since the underlying flagellar fibers were evident (Fig. 1a). Caput epididymal spermatozoa of Sepp1–/– males displayed additional flagellar defects that were not present in testicular spermatozoa and that also were not present in caput spermatozoa of wild-type animals (Fig. 1b–d). Most displayed a structural defect at the midpiece-principal piece junction that appeared to represent a missing segment of the mitochondrial sheath; many also displayed an abrupt flagellar angulation specifically at the junction of the midpiece and principal piece (Fig. 1c and d). This angulation was distinct from the midpiece bending occasionally observed at the level of the cytoplasmic droplet in caput spermatozoa of both wild-type and Sepp1–/– animals. Some caput spermatozoa of Sepp1–/– males also displayed unusual angulation at the head-tail junction or, more infrequently, a hairpin-like flagellar bend at the midpiece-principal piece junction (not shown). Cauda spermatozoa of Sepp1–/– mice demonstrated further maturation-dependent alterations in flagellar structure and a consistent flagellar phenotype. In nearly the entire sperm population, the flagellum was folded at the midpiece-principal junction into a sharp hairpin configuration (Fig. 1e). The occasional spermatozoon lacked the hairpin configuration and instead displayed a coiled flagellum or marked kinking at the head-tail and/or midpiece-principal piece junctions. The abnormal hairpin-fold of the sperm flagellum in Sepp1–/– animals was not a fixation artifact, and it was also seen in spermatozoa suspended in PBS or Tyrode solution. Although their motility was dramatically reduced compared with spermatozoa from wild-type controls, hairpin-configuration spermatozoa frequently displayed weak flagellar beating, particularly of the principal piece segment.



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  		FIG. 1. Phase-contrast photomicrographs showing developmental progression of sperm defects in Sepp1–/– mice. a) Testicular spermatozoa of null animals display an extended flagellum, but in some an abrupt narrowing of the posterior midpiece is evident (arrow). b) Caput spermatozoa from wild-type animals display normal flagellar architecture and no narrowing of the posterior midpiece (arrowhead). c and d) Caput spermatozoa from Sepp1–/ – animals typically show a narrowing of the posterior midpiece (arrow), and many display an abnormal flagellar bending (arrowhead) in this region of the flagellum. e) Most cauda spermatozoa of Sepp1–/– animals are sharply bent at the midpiece-principal piece junction (arrows) and exhibit a hairpin flagellar configuration

Electron microscopy identified a temporal development of lesions in specific flagellar organelles of spermatozoa from Sepp1–/– animals. In testicular spermatozoa from null animals, the cytoskeletal elements of the flagellum, including the axoneme, outer dense fibers, and fibrous sheath, appeared normal and identical to those of wild-type littermates (not shown). In contrast, many caput spermatozoa of null animals displayed altered geometric arrangements of these organelles (Fig. 2a). In cross-sections, the predominant midpiece abnormality was a peripheral accumulation of outer dense fibers and doublet microtubules in the cytoplasm between the mitochondrial sheath and plasma membrane. However, in most spermatozoa the outer dense fiber-axoneme complex, located within the mitochondrial sheath, appeared structurally normal. In the principal piece, cross-sections revealed a frequent absence of specific combinations of outer dense fibers-doublet microtubules Nos. 4–7, indicating that they represented the extruded fibers interposed between the midpiece mitochondrial sheath and plasma membrane. Cauda spermatozoa of null mice exhibited additional flagellar abnormalities. Most flagellar cross-sections revealed profiles of both the midpiece and principal piece contained within a common intact plasma membrane (Fig. 2b), a configuration that reflects the hairpin-bend observed by phase-contrast microscopy. The principal piece profiles typically lacked combinations of axonemal fibers Nos. 4–7, and these extruded fibers were apparent in the cytoplasm that surrounded the mitochondrial sheath and fibrous sheath. In addition to the alterations in flagellar cytoskeletal structures, specific abnormalities were also detected in the mitochondrial sheath of Sepp1–/– mice. Longitudinal sections of the midpiece-principal junction of caput spermatozoa from wild-type animals show that the wedge-shaped annulus maintains a close structural relationship with both the mitochondrial sheath and the fibrous sheath (Fig. 3a). In contrast, in caput spermatozoa of Sepp1–/– animals, the mitochondrial sheath terminated prematurely so that the annulus only associated with the fibrous sheath (Fig. 3b). A loss of flagellar integrity was frequently noted within the mitochondrial sheath-deficient zone of the posterior midpiece, and coincident with the abnormal flagellar bending, a fracturing of both the outer dense fibers and axonemal microtubules was observed. In cauda spermatozoa further mitochondrial abnormalities were detected, since some appeared pale, swollen, and lacking in the typical internal structural organization (Fig. 3b). These data demonstrate that a maturation-dependent development of specific lesions occurs in both the flagellar cytoskeletal elements and midpiece mitochondria of spermatozoa of Sepp1–/– mice.



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  		FIG. 2. a) Electron micrograph showing flagellar abnormalities in caput spermatozoa of Sepp1–/– animals. Some midpiece profiles exhibit supernumerary outer dense fibers and double microtubules in the peripheral cytoplasm, whereas the axoneme-outer dense fiber complex contained within the mitochondrial sheath appears intact. In cross-sections, some principal piece profiles showed a lack of specific axonemal doublet microtubules and their companion outer dense fiber (*principal piece lacks doublet microtubule No. 7). The plasma membrane appeared intact on all flagellar profiles. b) Electron micrograph of cauda spermatozoa of Sepp1–/– animal showing the profound flagellar abnormalities. In cross-sections of the anterior flagellum, both the midpiece and principal piece are contained within a common plasma membrane, an arrangement that reflects the hairpin flagellar configuration. Although the midpiece profiles display a normal arrangement of cytoskeletal elements, the principal piece profiles often lack specific doublet microtubules. Some of the midpiece mitochondria displays an electron dense matrix, whereas others (*) appear pale and swollen. Sections of the posterior flagellum reveal that many principal piece profiles lack specific doublet microtubules. df, Dense fibers; mt, microtubules; m, mitochondria; pp, principal piece; pm, plasma membrane; mp, midpiece



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  		FIG. 3. a) Electron micrograph showing the junction of the midpiece and principal piece segments of a caput spermatozoon from a wild-type male. Note that the wedge-shaped annulus adheres to both the fibrous sheath and the posterior-most mitochondria of the mitochondrial sheath. Note the normal appearance of the outer dense fibers. b) Electron micrograph showing the junction of the midpiece and principal piece segments of a caput spermatozoon from a Sepp1–/– male. The mitochondrial sheath is truncated so that its posterior-most mitochondria do not contact the annulus. The annulus remains at the junction of the midpiece and principal piece segments and is adherent to the anterior margin of the fibrous sheath. Note the disorganization and fracturing (arrow) of the outer dense fibers within the posterior midpiece. c) Electron micrograph of a proximal midpiece of a cauda spermatozoon of a Sepp1–/– male. Note the abnormally swollen and pale mitochondria (*) that occur intermittently along the mitochondrial sheath. a, Annulus; fs, fibrous sheath; m, mitochondria; df, dense fibers

Dietary Selenium Does Not Reverse Sperm Defects or Restore Fertility in Sepp1–/– Mice

Previously, several organs, including the testis, of Sepp1–/– animals were shown to contain low selenium levels [24, 25]. To test if selenium supplementation would restore normal sperm development and function, Sepp1–/– males were fed a diet that contained 1 ppm of selenium as sodium selenite beginning at weaning; spermatozoa were then examined at various time points between 2 and 12 mo of age. Spermatozoa from all these animals possessed the same maturation-dependent structural abnormalities as those from null animals fed a chow diet. Caput spermatozoa displayed the range of phenotypes described above and typically exhibited a truncated mitochondrial sheath, evident as a narrowing of the posterior midpiece, and variable flagellar angulations near the head-tail and/or midpiece-principal piece junctions (Fig. 4a). Nearly all cauda epididymal spermatozoa exhibited a hairpin flagellar configuration, and only an occasional spermatozoon displayed an extended flagellum (Fig. 4b and c). These data demonstrate that exogenous dietary selenium is insufficient to replace the role of SEPP1 in sperm development.



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  		FIG. 4. Differential interference contrast (a and b) and phase-contrast (c) images of spermatozoa from a wild-type male maintained on a selenium-supplemented diet. a) Caput spermatozoa show the same array of defects detected in the spermatozoa of Sepp1–/– animals fed a chow diet, including the gap in the posterior midpiece due to truncation of the mitochondrial sheath (brackets), abnormal flagellar bending at the midpiece-principal piece junction, and occasionally bending into a hairpin configuration flagellum (arrow). b and c) The flagella of most cauda spermatozoa are bent into a hairpin configuration and only occasional spermatozoa (arrows) display an extended flagellum

To test if higher amounts of dietary selenium reversed infertility of Sepp1–/– males, homozygous males fed a diet supplemented with 1 ppm of selenium were mated to wild-type females. Table 1 shows that compared with wild-type males on the same diet, Sepp1–/– males remained infertile. These data demonstrate that dietary selenium supplementation does not overcome an absolute requirement for SEPP1 in the maintenance of male fertility and suggest that SEPP1 may provide the physiologically relevant selenium source required for normal sperm development.


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  		TABLE 1. Comparison of Sepp1–/– and Sepp1+/+ male fertility

Selenium Deficiency Promotes Sperm Defects in C57B1/6 Mice Similar to Those in Sepp1–/– Males

If SEPP1 functions to provide the selenium required to support spermatogenesis, then selenium-deficient, wild-type Sepp1+/+ animals, which have dramatically reduced plasma SEPP1 levels [18, 44], should display a comparable temporal development of the same sperm flagellar defects as seen in Sepp1–/– animals. To test this possibility, C57B1/6 males were fed a selenium-deficient diet for up to 15 mo from the time of weaning. The measurement of liver and plasma glutathione peroxidase levels verified their selenium-deficient state by the 2-mo time point (Table 2). However, sperm abnormalities did not become evident until 4 mo of feeding the selenium-deficient diet. Spermatozoa from mice maintained on the selenium-deficient diet for 4 to 15 mo exhibited a similar temporal appearance of flagellar defects during spermiogenesis and after testicular development that appeared identical to those seen in Sepp1–/– mice. Testicular spermatozoa appeared normal except for a variable length gap in the posterior mitochondrial sheath (Fig. 5a). Caput epididymal spermatozoa also displayed the gap in the posterior mitochondrial sheath, and many spermatozoa showed various angulations of the head-tail and/or midpiece-principal piece junctions (Fig. 5b). A further striking alteration of flagellar structure was detected in cauda epididymal spermatozoa, and most possessed a hairpin bend at the midpiece-principal piece junction (Fig. 5c). Many spermatozoa with the hairpin flagellar bend also exhibited weak flagellar activity. Electron microscopic analysis of spermatozoa from the selenium-deficient animals confirmed the truncation of the posterior mitochondrial sheath and revealed that the annulus was attached to the fibrous sheath not the mitochondrial sheath (Fig. 5d). The abnormal bending of the flagellum at the posterior midpiece was also accompanied by the fracturing of outer dense fibers and axonemal microtubules (Fig. 5d). Ultrastructural analysis also confirmed that in hairpin-configuration flagella, the midpiece and principal piece are contained within a common plasma membrane (Fig. 5e). Furthermore, the axoneme and outer dense fibers within the midpiece retained a normal appearance, but various combinations of outer dense fiber-doublet microtubules Nos. 4– 7 were frequently extruded from the principal piece. Just as in the Sepp1–/– animals, these extruded fibers were present in the peripheral cytoplasm (Fig. 5e). These structural observations indicate that spermatozoa of Sepp1–/– and selenium-deficient Sepp1+/+ animals display similar structural changes during maturation in the epididymis.


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  		TABLE 2. Liver and plasma glutathione peroxidase activity (nmol/(ml x min)) of selenium-deficient and selenium-adequate C57Bl/6 mice.a



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  		FIG. 5. Phase-contrast (a–c) and electron micrographs (d and e) of spermatozoa from C57B1/6 mice maintained 6 mo on a selenium-deficient diet. a) Testicular spermatozoa possess an extended flagellum but frequently display an abrupt narrowing of the posterior midpiece (arrows). b) Caput spermatozoa also display a gap in the posterior mitochondrial sheath (arrows), and some exhibit an angulation of the flagellum. c) Cauda spermatozoa typically exhibit a hairpin bending of the flagellum at the midpiece-principal piece junction (arrows). d) Electron micrograph of midpiece-principal piece junction of spermatozoon in the process of flagellar bending; note the short gap between the posterior margin of the mitochondrial sheath and the annulus, which is affixed to the proximal end of the fibrous sheath. Some outer dense fibers at the site of flagellar bending are fractured. Note also that some midpiece mitochondria exhibit an electron dense appearance, whereas others (*) appear pale. e) Cross-section through hairpin bend region of a cauda sperm flagellum showing the midpiece and principal piece contained within a common plasma membrane. The axoneme-outer dense fiber complex of the midpiece but not the principal piece are intact, and extruded outer dense fibers and doublet microtubules are present within the peripheral cytoplasm. a, Annulus; fs, fibrous sheath; df, dense fibers; m, mitochondria; mp, midpiece; pp, principal piece; pm, plasma membrane; mt, microtubules

Testis Morphology in C57B1/6 and Sepp1–/– Mice

To identify histological abnormalities in the testes of Sepp1–/– or selenium-deficient C57B1/6 males, these testes were compared with those of C57B1/6 mice fed the 1-ppm selenium-supplemented diets. No detectable changes in the morphology of the Leydig cells or the seminiferous epithelium of the selenium-deficient or null animals were noted compared with normal controls (Fig. 6).



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  		FIG. 6. Photomicrograph showing representative seminiferous tubules from testes of a wild-type mouse (a), a Sepp1–/– mouse (b), and a wild-type mouse fed a selenium-deficient diet for 6 mo (c). No differences in structure of the seminiferous epithelium or interstitial cells were detected between the animals


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that spermatozoa from males with targeted deletion of the Sepp1 gene that are fed normal dietary levels of selenium progressively develop a set of structural defects during spermiogenesis and after testicular maturation that result in male infertility. Feeding supplemental dietary selenium to Sepp1–/– males does not reverse infertility or prevent the development of defective spermatozoa, indicating that SEPP1 plays an obligatory role in germ cell development. SEPP1 accounts for more than 60% of the selenium present in blood plasma [45], and, in Sepp1–/– males, selenium levels of blood plasma and the testis are reduced to ~6% and ~19% of wild-type males, respectively [25], indicating a likely role for SEPP1 in selenium delivery to the testis. It is noteworthy that the sperm defects detected in Sepp1–/– males appear similar to those seen in wild-type males fed a selenium-deficient diet. Since the blood plasma SEPP1 level of selenium-deficient animals is less that 10% of control males [18], a reduction in its level may be a key event in the development of the defective sperm phenotype just as in the Sepp1–/– male.

A SEPP1 trafficking pathway in the testis remains to be identified. In situ hybridization data demonstrate that Leydig cells, but not the seminiferous epithelium, express SEPP1 mRNA [38]. This finding indicates that SEPP1 required for germ cell development must be obtained from the testicular interstitial fluid, which contains liver-derived SEPP1 present in blood plasma and possibly SEPP1 produced locally by Leydig cells. We propose that the seminiferous tubule possesses both a specific uptake mechanism for SEPP1 and a degradation and delivery pathway to transport its selenium content to spermatids, where it is used for selenoprotein synthesis. SEPP1 is reported to bind membrane preparations, resulting in the formation of high-molecular-weight protein complexes [46]; however, a specific plasma membrane receptor for SEPP1 remains to be identified. In the seminiferous tubule only the peritubular cells, the Sertoli cells, and the spermatogonia are exposed to interstitial fluid and could function in SEPP1 binding. Since only the Sertoli cell is associated with the spermatid population, it appears most likely to function in transferring SEPP1-derived selenium to them. Both the selenoprotein degradation and biosynthesis pathways require specific sets of enzymes. When a selenoprotein such as selenoprotein P is degraded for use as a selenium source, selenocysteine ß-lyase is required to recover selenium from the released selenocysteine residues [26]. This enzyme is expressed in the testis, but its cell-specific distribution has not been examined. Likewise, selenophosphate synthetase is an obligatory enzyme for selenium utilization in selenoprotein biosynthesis [47, 48], and although it too is expressed in the testis [47], its cell-specific distribution pattern is not known. Future studies mapping the seminiferous epithelium for the cell-specific distribution of these key enzymes should provide insights into both the SEPP1 trafficking pathway and the molecular form of selenium delivered to spermatids.

Spermatids and spermatozoa from Sepp1–/– and from selenium-deficient wild-type males display a comparable temporal development of flagellar defects as a result of their SEPP1 and selenium-deficient status. In both models, the initial structural lesions are the assembly of a truncated mitochondrial sheath and atypical variations in mitochondrial shape. These mitochondrial sheath defects may reflect reduced spermatid expression of the selenoprotein phospholipid GPX4, the major structural protein [28] of the disulfide linked capsule [27], which defines sperm mitochondrial shape. In contrast, the flagellar cytoskeletal components form normally in spermatids of both the Sepp1–/– and the selenium-deficient males; however, in both models the axoneme-outer dense fiber complex becomes progressively disorganized as spermatozoa pass through the epididymis. This entails the selective sliding of doublet microtubules and outer dense fibers Nos. 4–7 from the lumen of the fibrous sheath and their coincident extrusion through the gap in the posterior mitochondrial sheath so that they become interposed between the mitochondrial sheath and plasma membrane. Ultimately, in most cauda spermatozoa, the flagellum becomes folded, specifically at the site of the truncation of the mitochondrial sheath, into a sharp hairpin configuration. There appear to be species variations in whether selenium deficiency results in loss of sperm viability. In the selenium-deficient rat, spermatozoa axonemal disintegration ruptures the plasma membrane, resulting in loss of viability [49], but in the mouse plasma membrane, integrity and viability are retained in many spermatozoa with the hairpin flagellar bend. The pattern of axonemal disintegration seen in spermatozoa of selenium-deficient animals also occurs in detergent-demembranated ATP-reactivated rat sperm models that also lack the mitochondrial sheath [50]. Moreover, examination of micrographs from many studies of sperm flagellar abnormalities in various mammals reveals identical patterns of axoneme disruption in epididymal spermatozoa, with a specific extrusion of outer dense fiber-doublet microtubule complexes Nos. 4–7 [50–54]. Whether defects in assembly of the mitochondrial sheath may also underlie this frequently encountered pattern of flagellar disintegration remains to be established. However, it is possible that identification of the molecular basis of flagellar defects in the Sepp1–/– and selenium-deficient models may provide insight into mechanisms underlying other cases of male infertility.

In spermatozoa of both Sepp1–/– and selenium-deficient wild-type mice, the flagellum becomes progressively disorganized as they transit through the epididymis. This finding suggests that the axonemal disintegration and the subsequent flagellar hairpin bend formation may be linked to development of sperm capacity for progressive motility, one of the signature events after testicular maturation [55]. Currently, it remains an open question whether the epididymis directly contributes to the development of the disorganized sperm phenotype. However, in addition to a proposed role in selenium transport, SEPP1 is also suggested to function in oxidant protection [21, 56], and it promotes the survival of cells in culture [57, 58]. We do not know whether SEPP1 is produced by the epididymis or is potentially transported to the tubule lumen, where it could directly interact with spermatozoa. Alternatively, epididymal selenoprotein synthesis could be compromised in the Sepp1–/– males, resulting in increased oxidative stress and reducing the protective capacity of the cauda luminal environment [55, 59]. Nonetheless, the present study demonstrates an absolute requirement for SEPP1 in sperm development and male fertility. Ongoing studies should reveal specific cellular and molecular roles for SEPP1 in spermiogenesis and after testicular sperm development.


   FOOTNOTES
 
1 Supported by National Institutes of Health grant HD-044863. Back

2 Correspondence: Gary E. Olson, Room T2208 MCN, 1161 21st Ave. S, Nashville, TN 37232-2175. FAX: 615 343 4539; gary.olson{at}vanderbilt.edu Back

Received: 26 January 2005.

First decision: 10 February 2005.

Accepted: 23 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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