Delivery of ferric ion to mouse spermatozoa is mediated by lipocalin internalization

The aim of this study was to illustrate the further process of 24p3 protein after association with epididymal spermatozoa. We have previously identified a caput-initiated 24p3 protein, which interacts with the spermatozoa surface in vitro. In the present study, we investigate another role of the 24p3 protein with spermatozoa. Mouse epididymal spermatozoa exhibit the ability to bind spontaneously with exogenous 24p3 protein, a part of which is further internalized into the spermatozoa in epididymal caput. We have now focused on this issue using freshly prepared spermatozoa from caudal region of epididymis. First, the cytosolic fractionation of spermatozoa has revealed that biotinylated 24p3 protein signal could be detected by supplying biotinylated protein under 37 degrees C incubation after 30 min at this experiment. Further, flow cytometric analysis of FITC-protein containing spermatozoa has revealed two distinct types of fluorescent spermatozoa, and microscopical experimentation with fluorescent FITC-24p3 protein has shown that the 24p3 protein did accumulate in the cytosolic portion of spermatozoa. All of these events, which showed protein uptake into the cell, demonstrated time- and temperature-dependence of endocytotic characteristics, these constituting the critical points in the process of endocytosis for spermatozoa as for other cells. Using a fluorometric method, the binding affinities of ferrous ion and ferric ion to 24p3 protein were shown to be (1.5+/-0.2)x10(6) and (3.0+/-0.4)x10(7)M(-1), respectively. We have also determined the internalization of this protein in the transition of iron into spermatozoa. We report here that spermatozoa, from the caudal epididymis, demonstrate the ability to bind with 24p3 protein and further internalize it and deliver the ferric ion to the spermatozoa via protein internalization. We suggest that the 24p3 protein plays a physiological role in spermatozoa in the context of protein-ligand complex internalization.     

Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology

To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes-lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)-with unknown functions in the epididymis. The novel gene, designated Serpina1f (serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except forLcp1. Testosterone (T) restored these reduced expressions, except forTeddm1; however, this restoration was not observed with 17 beta-estradiol (E2). Administration of T and E2 combination recovered the Serpina1f mRNA concentration; this recovery was also observed with T alone. However, the recovery of Cuzd1and Wfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed the Cuzd1, Wfdc16, and Serpina1f mRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.     

Expression of an endogenous murine leukemia virus-related proviral sequence is androgen regulated and primarily restricted to the epididymis/vas deferens and oviduct/uterus.

A cDNA representing a 5.2-kb defective, endogenous murine leukemia proviral sequence (EPI-EPS) was isolated from a C57BL/6 mouse cDNA epididymal library. Northern blot analysis demonstrated that EPI-EPS was predominantly expressed in the C57BL/6 mouse epididymis and vas deferens with 10-fold lower expression in the seminal vesicle, kidney, and submandibular gland. Analysis of tissues from other inbred strains of mice as well as the wild mouse, Mus musculus musculus, showed a similar pattern of tissue expression. EPI-EPS expression was also highly androgen regulated in both the reproductive and nonreproductive tissues of the C57BL/6 strain. However, a differential response to testosterone replacement was observed between tissues. Expression of EPI-EPS mRNA in the epididymis and vas deferens exhibited only a partial recovery to precastration levels after testosterone replacement; in the kidney and submandibular gland there was a complete recovery of EPI-EPS expression. Finally, EPI-EPS expression was also highly restricted in the female tissues, with expression limited to the oviduct and uterus. EPI-EPS, however, was not estrogen regulated in the female. These results suggest that a proviral sequence, EPI-EPS, is expressed in M. m. musculus and several inbred strains of mice due to its integration near a highly tissue-specific and androgen-regulated genetic locus.     

Molecular cloning of the cDNA for two major androgen-dependent secretory proteins of 18.5 kilodaltons synthesized by the rat epididymis

cDNA clones representing two closely related androgen-dependent secretory proteins of 18.5 kDa were selected by screening a rat epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the 18.5-kDa proteins. The entire amino acid sequence of the 18.5-kDa secretory proteins and a putative signal sequence of 18 amino acids was derived from 682 base pairs of the nucleotide sequence of overlapping cDNA clones. Confirmation of the identity of the cDNA clones was obtained by matching a partial amino acid sequence obtained for the N terminus of the pure protein with that of the sequence derived from the nucleotide code of the cDNA. Evidence is presented that the difference between the two closely related proteins may be associated with differential post-translational modification of the N terminus of the protein following cleavage of the signal sequence. Northern blot analysis revealed that the mRNA for the proteins is approximately 850 nucleotides long and that the concentration of the mRNA in the tissue is androgen-dependent. The proteins and their mRNAs were restricted to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from the skin, brain, liver, kidney, heart, skeletal muscle, and testis. With the exception of a weak cross-reaction with mouse epididymis, the proteins were not detected by Western blots of extracts of guinea pig, rabbit, or bull epididymis. The two proteins account for a substantial proportion of the total protein in epididymal luminal fluid and become incorporated as components of the sperm plasma membrane where they may play a specific role in the post-testicular phase of sperm development.     

IF3, a novel cell-differentiation factor,highly expressed in murine liver and ovary

The IF3 gene was isolated by expression cloning from a cDNA library of mouse oocytes. This gene was revealed to have no homology to any known gene and its cDNA encodes a 202-amino acid protein that contains a signal-peptide sequence. Moreover, an IF3 isoform, IF3(2), was expressed in both liver and ovary. Its cDNA encoded a 92-amino acid protein contains a signal-peptide sequence, which may be an alternative splice and frameshift form of IF3. The mRNA of IF3s was expressed in oocytes, ovary, and liver. Moreover, the gene expression of IF3s was regulated in a development-dependent manner in preimplantation-embryo and liver. Both IF3(1) and IF3(2) isoforms induced the differentiation of 2T3 and ATDC5 cells to the osteogenic and chondrogenic phenotype, respectively, suggesting that IF3s may modulate the differentiation status. Our findings suggest that IF3 may be one of the secreted factors that regulate oogenesis and certain liver functions.     

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.     

Molecular characterization of the mouse ribosomal protein S24 multigene family: a uniquely expressed intron-containing gene with cell-specific expression of three alternatively spliced mRNAs.

A family of 16 genes encoding the mouse ribosomal protein S24 was identified, and four members from this family were cloned. A single expressed intron-containing S24 gene (termed mrpS24) and one pseudogene (mrpS24p) were completely sequenced and characterized. The mrpS24 gene has seven exons and six introns spanning over 5.1 x 10(3) nucleotides (nt). The cap site of S24 was mapped to a G residue four nt upstream of a polypyrimidine tract and 15 nt downstream of a TATA-like (TATGA) element. The 5' region (-325 to +33) of the mrpS24 gene has a functional promoter that was able to express the fused chloramphenicol acetyltransferase (CAT) reporter gene. Two different forms of mouse S24 cDNA clones were previously isolated. Sequence analysis showed that one of these cDNA clones (termed S24a) lacks the entire exon V sequence (18 nt), and the deduced amino acid sequence is missing a C-terminal lysine residue encoded by the other cDNA (S24b). The pseudogene mrpS24p is flanked by an 11-bp direct repeat, and its sequence is almost identical to the S24 cDNA sequence, but it lacks two mini-exons, V and VI (20 nt), as in the cases of the human and rat S24 cDNAs. RT-PCR experiments demonstrated the existence of a third form (S24c) that similarly lacks both of the mini-exons, and suggested that different species of S24 mRNA might arise from alternative splicing of the mini-exons V and VI. Northern blot analysis showed that S24 expression is down- and up-regulated during adipocyte differentiation and in cellular transformation, respectively. RNase protection assays and RT-PCR experiments suggested that these cell-specific changes of S24 mRNA levels are mainly due to fluctuations in S24c mRNA level. Our results provide the first indication that a ribosomal protein gene is regulated by alternative usage of two mini-exons in a cell-specific manner.     

The major androgen-regulated secretory proteins of the rat epididymis bear sequence homology with members of the alpha 2u-globulin superfamily

The primary amino acid sequence of 18.5 kDa androgen-dependent secretory proteins of the rat epididymis has been compared with protein data-base sequences. The comparison has revealed that the epididymal proteins belong to the alpha 2u-globulin superfamily which includes human, rat and mouse urinary proteins, ungulate beta-lactoglobulins, and serum retinol-binding proteins. The homology suggests that the epididymal proteins may function to transport retinoids within the male reproductive tract. The androgen-dependent characteristics of the proteins and their tissue-specific nature have been ascertained by use of the protein's cDNA as a probe. The mRNA for the proteins was found in the epididymis of the rat and mouse, but not the epididymis of guinea-pig, rabbit, bull, boar, or ram.     

Molecular cloning of complementary deoxyribonucleic acid for an androgen-regulated epididymal protein: sequence homology with metalloproteins

Acidic epididymal glycoprotein (AEG) is a 31,000 molecular weight secretory protein of the rat epididymis. Screening of a rat epididymal cDNA library with affinity-purified AEG antiserum yielded cDNA for AEG. Identity of the clones was verified by comparison of amino acid sequence of the purified protein with the sequence derived from the nucleotide sequence of the cDNA isolates. Two classes of AEG cDNA, approximately 1500 base pairs (bp) and 950 bp in length, differed by 538 bp in the 3'-untranslated region and by four single nucleotide mismatches, one of which was in the coding region. Northern blot hybridization of epididymal RNA revealed two species of AEG mRNA, corresponding in length to each type of cDNA. Analysis of RNA from individual animals provided evidence that the two mRNA species are the products of allelic genes. In vivo studies demonstrated that the level of total AEG mRNA is regulated by androgen. Amino acid sequence homology of AEG with metal-binding domains of several proteins suggests that AEG is a metalloprotein.     

Next Generation Sequencing Analysis Reveals Segmental Patterns of microRNA Expression in Mouse Epididymal Epithelial Cells

The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract.     

Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation

In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin-related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage-specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII-VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation. © 1995 Wiley-Liss, Inc.     

Mouse cysteine-rich secretory protein 4 (CRISP4): a member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner

The final maturation of spermatozoa produced in the testis takes place during their passage through the epididymis. In this process, the proteins secreted into the epididymal lumen along with changes in the pH and salt composition of the epididymal fluid cause several biochemical changes and remodeling of the sperm plasma membrane. The Crisp family is a group of cysteine-rich secretory proteins that previously consisted of three members, one of which-CRISP1-is an epididymal protein shown to attach to the sperm surface in the epididymal lumen and to inhibit gamete membrane fusion. In the present paper, we introduce a new member of the Crisp protein family, CRISP4. The new gene was discovered through in silico analysis of the epididymal expressed sequence tag library deposited in the UniGene database. The peptide sequence of CRISP4 has a signal sequence suggesting that it is secreted into the epididymal lumen and might thus interact with sperm. Unlike the other members of the family, Crisp4 is located on chromosome 1 in a cluster of genes encoding for cysteine-rich proteins. Crisp4 is expressed in the mouse exclusively in epithelial cells of the epididymis in an androgen-dependent manner, and the expression of the gene starts at puberty along with the onset of sperm maturation. The identified murine CRISP4 peptide has high homology with human CRISP1, and the homology is higher than that between murine and human CRISP1, suggesting that CRISP4 represents the mouse counterpart of human CRISP1 and could have similar effects on sperm membrane as mouse and human CRISP1.