Two fibrinogen-like proteins, FGL1 and FGL2 are disulfide-linked subunits of oligomers that specifically bind nonviable spermatozoa.

Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments. The objective of the present study is to identify the subunit composition of the fgl2-containing oligomers both in the soluble and cocoon-like complex. Our proteomic studies indicate that the 260/280kDa oligomers (termed eFGL) contain two distinct disulfide-linked subunits; 64kDa fgl2 and 33kDa fgl1. Utilizing a PCR-based cloning strategy, the 33kDa polypeptide has been identified as fibrinogen-like protein-1 (fgl1). Immunocytochemical studies revealed that fgl1 selectively binds to defective spermatozoa in the cauda epididymidis. Northern blot analysis and in situ hybridization demonstrated the high expression of fgl1 in the principal cells of the proximal cauda epididymidis. Co-immunoprecipitation analyses of cauda epididymal fluid, using anti-fgl2, demonstrate that both fgl1 and fgl2 are present in the soluble eFGL. Our study is the first to show an association of fgl1 and fgl2 both in the soluble and in the sperm-associated eFGL. We conclude that our results provide new insights into the mechanisms by which the potentially unique epididymal protein functions in the recognition and elimination of defective spermatozoa.

Selenoprotein P is the major selenium transport protein in mouse milk.

Selenium is transferred from the mouse dam to its neonate via milk. Milk contains selenium in selenoprotein form as selenoprotein P (Sepp1) and glutathione peroxidase-3 (Gpx3) as well as in non-specific protein form as selenomethionine. Selenium is also present in milk in uncharacterized small-molecule form. We eliminated selenomethionine from the mice in these experiments by feeding a diet that contained sodium selenite as the source of selenium. Selenium-replete dams with deletion of Sepp1 or Gpx3 were studied to assess the effects of these genes on selenium transfer to the neonate. Sepp1 knockout caused a drop in milk selenium to 27% of the value in wild-type milk and a drop in selenium acquisition by the neonates to 35%. In addition to decreasing milk selenium by eliminating Sepp1, deletion of Sepp1 causes a decline in whole-body selenium, which likely also contributes to the decreased transfer of selenium to the neonate. Deletion of Gpx3 did not decrease milk selenium content or neonate selenium acquisition by measurable amounts. Thus, when the dam is fed selenium-adequate diet (0.25 mg selenium/kg diet), milk Sepp1 transfers a large amount of selenium to neonates but the transfer of selenium by Gpx3 is below detection by our methods.

Selenoprotein P and apolipoprotein E receptor-2 interact at the blood-brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration.

Selenoprotein P (Sepp1) and its receptor, apolipoprotein E receptor 2 (apoER2), account for brain retaining selenium better than other tissues. The primary sources of Sepp1 in plasma and brain are hepatocytes and astrocytes, respectively. ApoER2 is expressed in varying amounts by tissues; within the brain it is expressed primarily by neurons. Knockout of Sepp1 or apoER2 lowers brain selenium from ∼120 to ∼50 ng/g and leads to severe neurodegeneration and death in mild selenium deficiency. Interactions of Sepp1 and apoER2 that protect against this injury have not been characterized. We studied Sepp1, apoER2, and brain selenium in knockout mice. Immunocytochemistry showed that apoER2 mediates Sepp1 uptake at the blood-brain barrier. When Sepp1(-/-) or apoER2(-/-) mice developed severe neurodegeneration caused by mild selenium deficiency, brain selenium was ∼35 ng/g. In extreme selenium deficiency, however, brain selenium of ∼12 ng/g was tolerated when both Sepp1 and apoER2 were intact in the brain. These findings indicate that tandem Sepp1-apoER2 interactions supply selenium for maintenance of brain neurons. One interaction is at the blood-brain barrier, and the other is within the brain. We postulate that Sepp1 inside the blood-brain barrier is taken up by neurons via apoER2, concentrating brain selenium in them.

Sepp1(UF) forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1.

Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240-361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin(-/-) mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1(UF). Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1(UF) were compared with full-length Sepp1, Sepp1(Δ240-361), and Sepp1(U40S) as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1(U40S), which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H2O2 or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1(UF) from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1(UF) to function by itself as a peroxidase. Ultimately, plasma Sepp1(UF) and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3.

Maternal-fetal transfer of selenium in the mouse.

Selenoprotein P (Sepp1) is taken up by receptor-mediated endocytosis for its selenium. The other extracellular selenoprotein, glutathione peroxidase-3 (Gpx3), has not been shown to transport selenium. Mice with genetic alterations of Sepp1, the Sepp1 receptors apolipoprotein E receptor-2 (apoER2) and megalin, and Gpx3 were used to investigate maternal-fetal selenium transfer. Immunocytochemistry (ICC) showed receptor-independent uptake of Sepp1 and Gpx3 in the same vesicles of d-13 visceral yolk sac cells, suggesting uptake by pinocytosis. ICC also showed apoER2-mediated uptake of maternal Sepp1 in the d-18 placenta. Thus, two selenoprotein-dependent maternal-fetal selenium transfer mechanisms were identified. Selenium was quantified in d-18 fetuses with the mechanisms disrupted. Maternal Sepp1 deletion, which lowers maternal whole-body selenium, decreased fetal selenium under selenium-adequate conditions but deletion of fetal apoER2 did not. Fetal apoER2 deletion did decrease fetal selenium, by 51%, under selenium-deficient conditions, verifying function of the placental Sepp1-apoER2 mechanism. Maternal Gpx3 deletion decreased fetal selenium, by 13%, but only under selenium-deficient conditions. These findings indicate that the selenoprotein uptake mechanisms ensure selenium transfer to the fetus under selenium-deficient conditions. The failure of their disruptions (apoER2 deletion, Gpx3 deletion) to affect fetal selenium under selenium-adequate conditions indicates the existence of an additional maternal-fetal selenium transfer mechanism.

Production of selenoprotein P (Sepp1) by hepatocytes is central to selenium homeostasis.

BACKGROUND: Sepp1 transports selenium, but its complete role in selenium homeostasis is not known. RESULTS: Deletion of Sepp1 in hepatocytes increases liver selenium at the expense of other tissues and decreases whole-body selenium by increasing excretion. CONCLUSION: Sepp1 production by hepatocytes retains selenium in the organism and distributes it from the liver to peripheral tissues. SIGNIFICANCE: Sepp1 is central to selenium homeostasis. Sepp1 is a widely expressed extracellular protein that in humans and mice contains 10 selenocysteine residues in its primary structure. Extra-hepatic tissues take up plasma Sepp1 for its selenium via apolipoprotein E receptor-2 (apoER2)-mediated endocytosis. The role of Sepp1 in the transport of selenium from liver, a rich source of the element, to peripheral tissues was studied using mice with selective deletion of Sepp1 in hepatocytes (Sepp1(c/c)/alb-cre(+/-) mice). Deletion of Sepp1 in hepatocytes lowered plasma Sepp1 concentration to 10% of that in Sepp1(c/c) mice (controls) and increased urinary selenium excretion, decreasing whole-body and tissue selenium concentrations. Under selenium-deficient conditions, Sepp1(c/c)/alb-cre(+/-) mice accumulated selenium in the liver at the expense of extra-hepatic tissues, severely worsening clinical manifestations of dietary selenium deficiency. These findings are consistent with there being competition for metabolically available hepatocyte selenium between the synthesis of selenoproteins and the synthesis of selenium excretory metabolites. In addition, selenium deficiency down-regulated the mRNA of the most abundant hepatic selenoprotein, glutathione peroxidase-1 (Gpx1), to 15% of the selenium-replete value, while reducing Sepp1 mRNA, the most abundant hepatic selenoprotein mRNA, only to 61%. This strongly suggests that Sepp1 synthesis is favored in the liver over Gpx1 synthesis when selenium supply is limited, directing hepatocyte selenium to peripheral tissues in selenium deficiency. We conclude that production of Sepp1 by hepatocytes is central to selenium homeostasis in the organism because it promotes retention of selenium in the body and effects selenium distribution from the liver to extra-hepatic tissues, especially under selenium-deficient conditions.

Glutathione peroxidase-3 produced by the kidney binds to a population of basement membranes in the gastrointestinal tract and in other tissues.

Glutathione peroxidase-3 (Gpx3), the extracellular glutathione peroxidase synthesized largely in the kidney, binds to basement membranes of renal cortical epithelial cells. The present study assessed extrarenal expression of Gpx3 using RT-PCR and presence of Gpx3 protein using immunocytochemistry. Gpx3 expression was higher in kidney and epididymis than in other tissues. Gpx3 bound to basement membranes of epithelial cells in the gastrointestinal tract, the efferent ducts connecting the seminiferous tubules with the epididymis, the bronchi, and type II pneumocytes. It was not detected on the basement membrane of type I pneumocytes. Gpx3 was also present in the lumen of the epididymis. Transplantation of Gpx3(+/+) kidneys into Gpx3(-/-) mice led to Gpx3 binding to the same basement membranes to which it bound in Gpx3(+/+) mice but not to its presence in the epididymal lumen. These results show that Gpx3 from the blood binds to basement membranes of specific epithelial cells and indicate that the cells modify their basement membranes to cause the binding. They further indicate that at least two Gpx3 compartments exist in the organism. In one compartment, kidney supplies Gpx3 through the blood to specific basement membranes in a number of tissues. In the other compartment, the epididymis provides Gpx3 to its own lumen. Tissues other than kidney and epididymis express Gpx3 at lower levels and may supply Gpx3 to other compartments.

Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells.

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.

Megalin mediates selenoprotein P uptake by kidney proximal tubule epithelial cells.

Selenoprotein P (Sepp1) contains most of the selenium in blood plasma, and it is utilized by the kidney, brain, and testis as a selenium source for selenoprotein synthesis. We recently demonstrated that apolipoprotein E receptor-2 (ApoER2) is required for Sepp1 uptake by the testis and that deletion of ApoER2 reduces testis and brain, but not kidney, selenium levels. This study examined the kidney Sepp1 uptake pathway. Immunolocalization experiments demonstrated that Sepp1 passed into the glomerular filtrate and was specifically taken up by proximal tubule epithelial cells. Neither the C terminus selenocysteine-rich domain of Sepp1 nor ApoER2 was required for Sepp1 uptake by proximal tubules. Tissue ligand binding assays using cryosections of Sepp1-/- kidneys revealed that the proximal tubule epithelium contained Sepp1-binding sites that were blocked by the receptor-associated protein, RAP, an inhibitor of lipoprotein receptor-ligand interactions. Ligand blotting assays of kidney membrane preparations fractionated by SDS-PAGE revealed that Sepp1 binds megalin, a lipoprotein receptor localized to the proximal tubule epithelium. Immunolocalization analyses confirmed the in vivo co-localization of Sepp1 and megalin in wild type kidneys and demonstrated the absence of proximal tubule Sepp1 uptake in megalin null mice. These results demonstrate that kidney selenium homeostasis is mediated by a megalin-dependent Sepp1 uptake pathway in the proximal tubule.

A role for the inositol kinase Ipk1 in ciliary beating and length maintenance.

Cilia project from cells as membranous extensions, with microtubule structural cores assembling from basal bodies by intraflagellar transport (IFT). Here, we report a ciliary role for the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1) that generates inositol hexakisphosphate. In zebrafish embryos, reducing Ipk1 levels inhibited ciliary beating in Kupffer's vesicle and decreased ciliary length in the spinal canal, pronephric ducts, and Kupffer's vesicle. Electron microscopy showed that ciliary axonemal structures were not grossly altered. However, coincident knockdown of Ipk1 and IFT88 or IFT57 had synergistic perturbations. With GFP-Ipk1 enriched in centrosomes and basal bodies, we propose that Ipk1 plays a previously uncharacterized role in ciliary function.

Deletion of apolipoprotein E receptor-2 in mice lowers brain selenium and causes severe neurological dysfunction and death when a low-selenium diet is fed.

Selenoprotein P (Sepp1) is a plasma and extracellular protein that is rich in selenium. Deletion of Sepp1 results in sharp decreases of selenium levels in the brain and testis with dysfunction of those organs. Deletion of Sepp1 also causes increased urinary selenium excretion, leading to moderate depletion of whole-body selenium. The lipoprotein receptor apolipoprotein E receptor-2 (apoER2) binds Sepp1 and facilitates its uptake by Sertoli cells, thus providing selenium for spermatogenesis. Experiments were performed to assess the effect of apoER2 on the concentration and function of selenium in the brain and on whole-body selenium. ApoER2-/- and apoER2+/+ male mice were fed a semipurified diet with selenite added as the source of selenium. ApoER2-/- mice had depressed brain and testis selenium, but normal levels in liver, kidney, muscle, and the whole body. Feeding a selenium-deficient diet to apoER2-/- mice led to neurological dysfunction and death, with some of the characteristics exhibited by Sepp1-/- mice fed the same diet. Thus, although it does not affect whole-body selenium, apoER2 is necessary for maintenance of brain selenium and for prevention of neurological dysfunction and death under conditions of selenium deficiency, suggesting an interaction of apoER2 with Sepp1 in the brain.

Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis.

Selenium is a micronutrient that is essential for the production of normal spermatozoa. The selenium-rich plasma protein selenoprotein P (Sepp1) is required for maintenance of testis selenium and for fertility of the male mouse. Sepp1 trafficking in the seminiferous epithelium was studied using conventional methods and mice with gene deletions. Immunocytochemistry demonstrated that Sepp1 is present in vesicle-like structures in the basal region of Sertoli cells, suggesting that the protein is taken up intact. Sepp1 affinity chromatography of a testicular extract followed by mass spectrometry-based identification of bound proteins identified apolipoprotein E receptor 2 (ApoER2) as a candidate testis Sepp1 receptor. In situ hybridization analysis identified Sertoli cells as the only cell type in the seminiferous epithelium with detectable ApoER2 expression. Testis selenium levels in apoER2(-/-) males were sharply reduced from those in apoER2(+/+) males and were comparable with the depressed levels found in Sepp1(-/-) males. However, liver selenium levels were unchanged by deletion of apoER2. Immunocytochemistry did not detect Sepp1 in the Sertoli cells of apoER2(-/-) males, consistent with a defect in the receptor-mediated Sepp1 uptake pathway. Phase contrast microscopy revealed identical sperm defects in apoER2(-/-) and Sepp1(-/-) mice. Co-immunoprecipitation analysis demonstrated an interaction of testis ApoER2 with Sepp1. These data demonstrate that Sertoli cell ApoER2 is a Sepp1 receptor and a component of the selenium delivery pathway to spermatogenic cells.

Demonstration of ubiquitin thiolester formation of UBE2Q2 (UBCi), a novel ubiquitin-conjugating enzyme with implantation site-specific expression.

We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin-conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular mass, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin-conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendrogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes, and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The Km for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6(3/4) pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of Day 8 pregnant animals. No expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the Day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression, with low-to-undetectable levels in implantation sites of Day 6(3/4) pregnant endometrium but high levels in luminal epithelial cells of Day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.

Identification of a hamster sperm 26-kilodalton dehydrogenase/reductase that is exclusively localized to the mitochondria of the flagellum.

Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.

Selenoprotein P is required for mouse sperm development.

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.

Tyrosine phosphorylation generates multiple isoforms of the mitochondrial capsule protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), during hamster sperm capacitation.

Sperm capacitation is a maturation process, occurring in the female reproductive tract, that produces fertilization-competent spermatozoa. Protein tyrosine phosphorylation represents an important event in capacitation. The present study demonstrates the capacitation-dependent tyrosine-phosphorylation of phospholipid hydroperoxide glutathione peroxidase (PHGPx), the disulfide cross-linked, major structural protein of the sperm mitochondrial capsule. Immunofluorescence microscopy using an antiphosphotyrosine monoclonal antibody (anti-pY20) demonstrated the presence of capacitation-associated tyrosine phosphorylated proteins in the flagellum of hamster spermatozoa. Among the tyrosine-phosphorylated polypeptides (M(r) 19,000- 99,000), a 19-kDa polypeptide was the only one that can be solubilized completely by Triton X-100-dithiothreitol (DTT). The 19-kDa polypeptide was purified by anion-exchange chromatography and by immunoaffinity chromatography. Proteomic identification of the 19-kDa polypeptide by nano-electrospray tandem mass spectrometry yielded six peptides that matched the National Center for Biotechnology Information (NCBI) database sequences of bovine PHGPx. Indirect immunofluorescence localized PHGPx to the midpiece of the flagellum and the immunoblot analysis demonstrated its DTT-dependent release from purified flagella. DTT extracts of noncapacitated spermatozoa exhibited a charge train of four major PHGPx isoforms (pIs 7.5- 9.0) by two-dimensional PAGE, whereas capacitated spermatozoa revealed the generation of new acidic PHGPx isoforms with isoelectric points ranging between pH 6.0-7.0 and 4.0-5.0, indicating that it is posttranslationally modified during capacitation. These data suggest that the tyrosine-phosphorylation of PHGPx may represent an important event in the signaling pathway(s) associated with capacitation and could potentially affect mitochondrial function.

Region-specific expression and secretion of the fibrinogen-related protein, fgl2, by epithelial cells of the hamster epididymis and its role in disposal of defective spermatozoa.

The cauda epididymidis functions in the storage and protection of mature, fertile spermatozoa. We previously identified a region-specific secretory glycoprotein (termed HEP64) of the hamster proximal cauda epididymidis that specifically bound and coated the nonviable, but not the viable, spermatozoa within the epididymal lumen. In this study we employed expression screening of a hamster epididymal cDNA library to obtain the full-length sequence of HEP64 and to identify it as the fibrinogen-like protein fgl2. Northern blot analysis demonstrated that fgl2 mRNA is highly expressed by the proximal cauda epididymidis in comparison to other hamster tissues examined, and, in situ hybridization analysis of the epididymis revealed that fgl2 mRNA exhibited a region- and principal cell-specific expression pattern. Immunohistochemistry confirmed the association of fgl2 with abnormal spermatozoa in the cauda epididymidis and revealed smaller fgl2-containing particles. Immunoelectron microscopy revealed that fgl2 was distributed throughout an amorphous, "death cocoon," complex assembled onto abnormal spermatozoa and that the smaller fgl2 aggregates consisted of the amorphous material with embedded sperm fragments, organelles, and membrane vesicles. A protocol was developed to isolate an enriched death cocoon fraction. SDS-PAGE and microsequence analyses revealed that the Mr 64,000 fgl2 monomer was assembled into two disulfide-linked oligomers of Mr 260,000 and 280,000. These data demonstrate that the epididymis possesses a specific mechanism to identify and envelop defective spermatozoa with a protein complex containing the fibrinogen-like protein fgl2. We propose that this represents an important protective mechanism not only to shield the viable sperm population from potentially deleterious enzymes released by dying spermatozoa but also to prevent the release of sperm proteins that could initiate an immune response if they escaped the epididymal environment.

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis.

Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.

Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats.

In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.

Differential expression of genes in the endometrium at implantation: upregulation of a novel member of the E2 class of ubiquitin-conjugating enzymes.

The process of embryo attachment and implantation is accompanied by dramatic cellular and functional changes in the endometrium, the control and mechanisms of which are not clearly understood. The cDNA cloning of differentially expressed genes, specifically at implantation sites in the rabbit endometrium, was used to identify genes controlling functional and remodeling changes. Tissue from the endometrium of Day 6(3/4) (preimplantation) and Day 8 (implantation initiation) pregnant rabbits was used to screen for differentially expressed genes by combined cDNA subtraction/suppressive hybridization. Twenty-nine differentially expressed genes were identified encoding protein modification enzymes, signaling proteins, structural proteins, and enzymes. One of these is a novel member of the E2 ubiquitin-conjugating enzyme family we have designated UBCi (i for implantation), which displayed dramatic nucleotide and deduced amino acid sequence conservation between rabbits, humans, and mice. In situ hybridization indicated UBCi expression exclusively in the luminal epithelium of the endometrium while glandular epithelium, trophoblast, and myometrium were negative. Expression was specific for epithelial cells at implantation sites and was not detected in non-implant-site endometrium. UBCi mRNA was detected in both the mesometrial and antimesometrial epithelial cells of the implantation sites, sites undergoing both differentiation and/or apoptosis. These results identify a group of differentially expressed genes in the endometrium including UBCi and provide new focal targets for studying processes controlling cellular remodeling during implantation. The important roles of ubiquitination in controlling the activities and turnover of key signaling proteins suggest potential roles in controlling critical aspects of implantation.