Gene encoding a mammalian epididymal protein.

Polyclonal antibodies raised against a 20 kD epididymal protein (EP20) were used to isolate the cDNA from a human testis lambda gt11 expression library. The nucleotide sequence of the cDNA consisted of 1908 base pairs (bp) containing an open reading frame composed of 1479 bp encoding a polypeptide of 493 amino acid residues. The nucleotide sequence of EP-20 cDNA had 97% identities (282/288) with ESTO 0991 Homo Sapiens cDNA clone HHC M14 in the reverse orientation. The HHC M14 sequence corresponded to a segment in the non-translatable 3'end of EP-20 cDNA. The amino acid sequence of the deduced polypeptide showed no homology with reported polypeptides. The epididymal protein may be involved in sperm maturation and/or capacitation.

Testin secreted by Sertoli cells is associated with the cell surface, and its expression correlates with the disruption of Sertoli-germ cell junctions but not the inter-Sertoli tight junction.

Testin is a testosterone-responsive Sertoli cell secretory product. In the present study, we demonstrated that the amount of testin secreted by Sertoli cells in vitro was comparable with several other Sertoli cell secretory products. However, virtually no testin was found in the luminal fluid and cytosols of the testis and epididymis when the intercellular junctions were not previously disrupted, suggesting that secreted testin may be reabsorbed by testicular cells in vivo. Studies using Sertoli cells with and without a cell surface cross-linker and radioiodination in conjunction with immunoprecipitation illustrated the presence of two polypeptides of 28 and 45 kDa, which constitute a binding protein complex that anchors testin onto the cell surface. The 28- and 45-kDa peptide appear to be residing on and inside the cell surface, respectively. Immunogold EM studies illustrated testin was abundantly localized on the Sertoli cell side of the ectoplasmic specialization (a modified adherens junction) surrounding developing spermatids. In contrast, very few testin gold particles were found at the site of inter-Sertoli tight junctions. When the inter-Sertoli tight junctions were formed or disrupted, no significant change in testin expression was noted. This is in sharp contrast to the disruption of Sertoli-germ cell junctions, which is accompanied by a surge in testin expression. These results demonstrate the usefulness of testin in examining Sertoli-germ cell interactions.

Testins are localized to the junctional complexes of rat Sertoli and epididymal cells.

UNLABELLED: Testin I and Testin II were originally identified as Sertoli cell products with similar NH2-terminal amino acid sequences. Secretion of testins is stimulated by testosterone in Sertoli cell-enriched cultures. By contrast the secretion of testins from intact seminiferous tubules appears to be inversely related to germ cell number. In the present study testin antiserum that recognized both Testin I and Testin II ("testin") was used to localize these proteins in tissue secretions by immunofluorescence. Testin was localized at the base of the seminiferous epithelium at Sertoli-Sertoli junctions. Fluorescence also appeared to be located at the sites of interaction between spermatoids and Sertoli cells. A punctate pattern of fluorescence was also present in the cytoplasm of Leydig cells; without electron microscopic studies it was not possible to determine which structures the antibodies bound to in these cells. In the epididymis the reaction product was localized at the apices of the epithelial cells adjacent to the lumen at the sites of known junctional complexes. A variety of positive and negative controls indicated that staining was specific for testins. CONCLUSIONS: This is the first study to associate testins with junctional complexes. Relative to other junctional proteins, testins are unusual because of their small size and because they are secreted proteins.

Expression of the calpastatin gene segment during spermiogenesis in human testis: an in situ hybridization study.

Serum obtained from an infertile woman contained antibodies that agglutinate human sperm. The antibodies interacted with a sperm protein with an estimated M(r) of 17.5 kD. The cDNA coding the 17.5-kD protein was isolated from a human testis lambda gt11 expression library and identified as a segment of the calpastatin gene. Single-stranded 35S-labeled RNA probes were prepared from the calpastatin cDNA segment. Using the techniques of in situ hybridization, the calpastatin mRNA was located in spermatids of human testis. The results support a previous observation that the calpastatin segment is produced during spermiogenesis and suggest that transcription of the calpastatin gene occurred during the postmeiotic haploid stage of spermatogenesis.

Rat testin is a newly identified component of the junctional complexes in various tissues whose mRNA is predominantly expressed in the testis and ovary.

Testin I and testin II are the two molecular variants of testin that are synthesized and secreted by Sertoli cells in vitro. N-Terminal and partial internal amino acid sequence analysis of testin I and testin II reveals that these molecules are identical with the exception that testin II has three extra N-terminal amino acids of TAP compared to testin I. Studies using immunohistochemistry suggested that testin is a component of the specialized junctional complexes in the seminiferous epithelium and other tissues. Immunoreactive testin is localized not only at Sertoli-Sertoli and Sertoli-germ cell junctions, but also at sites of similar junctions in the liver, epididymis, kidney, and intestine. Other physiological studies have shown that the secretion of testin is tightly coupled to the presence of germ cells. In view of its possible role in germ cell development and its unique localization in the cell junction, the purpose of the present study was to determine the structure of testin by sequencing its full-length cDNA. Two synthetic degenerate oligonucleotides based on the N-terminal and an internal amino acid sequence were used for polymerase chain reaction (PCR) to obtain a 289-bp cDNA fragment. This PCR product was subsequently used to isolate a 1371-bp cDNA from a cDNA expression library constructed from Sertoli cell poly(A) RNA. This cDNA coded for a 333 amino acid peptide that starts with an ATG initiation codon from the 5' end and ends with a TGA termination codon located 245 nucleotides before the polyadenylation site. The deduced amino acid sequence indicates that testin contains a 16 amino acid signal peptide with two possible cleavage sites that yield 314 and 317 amino acids for testin I and testin II with calculated molecular weights of 36,029 and 36,299, respectively. Comparison of the entire coding region of testin with existing sequences at Genbank, EMBL, and Protein Identification Resource indicates that testin shares 58%, 57.4%, and 61% identity with rat, mouse, and human cathepsin L at the amino acid level, respectively. The positions of all of the 7 Cys residues and 8 of the 10 Trp residues in testin are conserved with respect to those present in cathepsin L. It is noted that Cys-122 in the predicted active site of cathepsin L was replaced with Ser-122 in testin. In view of the striking primary sequence homology between testin and cathepsin L, we assayed the proteolytic activity of testin using conditions known to activate cathepsin L.(ABSTRACT TRUNCATED AT 400 WORDS)

Fertility studies with antisperm antibodies.

Serum obtained from an infertile subject possessed antibodies that interacted with a human sperm glycoprotein with an estimated M(r) of 17,550 and pI of 5.65 containing 17.7% neutral hexoses and designated as the BS-17 component. Polyclonal antibodies raised against the BS-17 antigen blocked the capacity of human sperm to fertilize zona-free hamster ova in vitro; however, the antibodies did not influence the binding of human sperm to zone-free ova or alter the motility of human sperm. The antibodies inhibited the capacity of mouse sperm to fertilize ova upon in vivo insemination. The BS-17 antigen was detected in human, rat, mouse, rabbit, and hamster sperm by an immunocytochemical method, using polyclonal anti-BS-17 antibodies. Intense staining occurred over the surface of the acrosomal region of all mammalian sperm. The results suggest that the production of anti-BS-17 antibodies contribute to infertility by preventing the capacitation of sperm and/or by blocking the ability of capacitated sperm to fertilize the egg.

Cyclic and postnatal developmental changes of testin in the rat seminiferous epithelium--an immunohistochemical study.

Testin is an authentic Sertoli cell secretory protein consisting of two molecular variants designated testin I (M(r) 35 000) and testin II (M(r) 37 000). N-Terminal amino acid sequence analysis revealed that testin I is identical to testin II except that testin II has three extra N-terminal amino acids of threonine-alanine-proline (TAP). Earlier studies by immunoflorescence microscopy have shown that testin is detected in the seminiferous epithelium consistent with localization in the junctions between Sertoli cells as well as Sertoli-germ cells, and that it appears to be a component of junctional complexes in the testis. In the present study, we have examined the localization of testin in different stages of the spermatogenic cycle of the adult rat testis when germ cells migrate from the basal portion of the seminiferous epithelium to the tubular lumen. In stages I-IV, testin was localized mainly in the basement laminae in the junctional complexes between adjacent Sertoli cells as well as between Sertoli cells, spermatogonia, and pachytene spermatocytes. When elongated spermatids were embedded into the seminiferous epithelium in stage VII of the cycle, testin was detected predominantly on the concave side of the elongated spermatids, but relatively few testin reaction products were seen in the round spermatids. In the beginning of stage VIII of the spermatogenic cycle, intense testin immunoreactive substances were detected around the heads of the elongated spermatids; these substances were virtually undetectable in late stage VIII after the release of the mature sperm into the tubular lumen, suggesting that testin may be a novel marker to divide stage VIII into stages VIIIa and VIIIb.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development.

BACKGROUND: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. METHODS: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. RESULTS: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. CONCLUSIONS: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.

Eliciting an immune response by plasmid DNA encoding a human sperm protein (HSD-1).

The cDNA encoding a human sperm membrane designated as HSD-1 was isolated from a human testis lambda gt11 cDNA expression library and assigned the accession number U12978 by GenBank. HSD-1 was conjugated to an eukaryotic expression plasmid (pRSV) to construct the recombinant plasmid pRSV-HSD-1. Female mice were inoculated intramuscularly with the plasmid DNA and the expression of HSD-1 was determined. HSD-1 mRNAs were detected in myocytes and endomysial connective tissue cells of the quadriceps muscle by in situ hybridization. Spleen of inoculated animals contained an increased number of cytotoxic T lymphocytes, phagocytes, and plasma cells. Fertility of the treated animals was not affected. Thus, intramuscular inoculation of female mice with the plasmid DNA (pRSV-HSD-1) results in the expression of HSD-1 and may elicit a tissue-mediated immune response.

Effect of gossypol on the potential difference of rat seminiferous tubules.

The p.d. of the rat seminiferous tubules was 4.75 +/- 1.39 mV (lumen negative) at 35 degrees C and varied linearly with temperatures from 26 to 40 degrees C. A depolarization of the seminiferous tubules was found in the rats administered with gossypol at the dosage of 30 mg/kg body weight for 3 weeks, the p.d. lowered to 3.63 +/- 0.79 mV at 35 degrees C and was independent of the changes of seminiferous tubules temperature. In the 5-week-gossypol-treatment group, the tracer penetrated not only the myoid cell layer, but also went beyond the tight junction complexes between Sertoli cells. The lanthanum appeared in the cleft surrounding spermatogonia. In the 8-week-gossypol-treatment group, the lanthanum was found in the adluminal compartment. It indicates that gossypol can cause a dysfunction of the Sertoli cells and blood-testis barrier and disturb a good physiological environment for the developing spermatocytes.

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.

Molecular cloning and characterization of a novel testis-specific nucleoporin-related gene.

A 20-kDa sperm membrane protein cDNA, designated as RSD-1, was isolated by epitope selection from a rat testis lambda gtll expression library. RSD-1 was used as a probe to screen a human testis lambda ZAPII cDNA expression library. A cDNA designated as BS-63 was isolated and found to consist of 1933 bp with an open reading frame of 1824 bp and assigned the accession number U64675 by GenBank. The deduced polypeptide consisted of 608 amino acid residues containing XFXFG or FG motifs that are characteristic of nuclear pore complex (NPC) proteins and act as potential binding sites for Ran. The N-terminal region has high homology with RanBP2/Nup358, a nucleoporin component, showing that BS-63 is a member of the NPC family. Northern blot analysis of mRNAs prepared from various human tissues shows that BS-63 is transcribed in two forms: 6.0 and 8.5 kb. The 8.5-kb transcript was present in low amounts in several somatic tissues, whereas the 6.0-kb transcript is expressed only in testis. In situ hybridization analysis of human testis sections showed that BS-63 mRNA is expressed only in germ cells at all stages of spermatogenesis. Sertoli cells did not transcribe the gene.

Expression of the BE-20 epididymal protein gene: in situ hybridization.

A protein designated as BE-20 was purified from cauda epididymal fluid of male rabbits and the amino acid sequence of the N-terminus was determined. A 23-mer oligonucleotide coding the N-terminal eight amino acids of the BE-20 protein was synthesized. The oligonucleotide was used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. Digoxigenin-labeled BE-20 cDNA was prepared and used as a hybridization probe to detect the specific mRNA. The probe interacted with a 1.2-kb mRNA prepared from rabbit epididymis; mRNAs prepared from rabbit testis gave negative reaction. Using tissue sections, the BE-20 mRNA was located in the epithelial cells of the cauda epididymis and proximal segment of the ductus deferens by in situ hybridization method. Sections of the corpus and caput epididymis, testis, and liver gave negative reaction. Polyclonal anti-BE-20 antibodies were raised and found to inhibit in vitro the capacity of human sperm to penetrate zona-free hamster ova. The results suggest that BE-20 protein may influence maturation of spermatozoa during its movement through the epididymis and/or the capacity of sperm to fertilize ova.

Gene encoding a human testis Sertoli cell component related to LIM domain protein.

The cDNA (HED-2) encoding a 20 kDa protein found in mammalian epididymal fluid was isolated from a human testis expression library. It is composed of 1908 bp, containing a reading frame of 1479 bp, coding a polypeptide consisting of 493 amino acids, and assigned the accession number: U15158 by GenBank (Biochem. Mol. Biol. Int. 34, 1131-1136, 1994). HED-2 has 99% identity with the zyxin gene in amino acid sequence, a component of cell junction matrix and a member of the LIM domain protein family. Northern blot analysis of RNAs prepared from various human tissues showed that the HED-2 gene was expressed in all tissues analyzed. Sertoli cells of human testis expressed the gene as determined by an in situ hybridization method. The present study shows that the HED-2 gene is a member of the LIM domain protein family.

Human sperm membrane protein (hSMP-1): a developmental testis-specific component during germ cell differentiation.

Serum was obtained from an infertile woman having antibodies with sperm agglutinating activity. The antibodies interacted with a human sperm membrane protein (hSMP-1) with an estimated Mr of 55 kD. The gene (HSD-1) coding hSMP-1 was isolated from a human testis cDNA expression library and assigned the accession number U12978. The cDNA was conjugated to a prokaryotic expression vector to construct the recombinant vector, pRSET-HSD-I, which was expressed in Escherichia coli. The recombinant hSMP-1 was isolated and used to immunize rabbits to raise polyclonal antibodies. Usingan immunocytochemical technique, hSMP-1 protein was immunolocalized in germ cells of human testis at all stages of spermatogenesis. mRNAs were prepared from 16 different human tissues and analyzed by Northern blot using HSD-1 as probe. A positive reaction was elicited only with testis mRNA. The present findings suggest that the expression of hSMP-1 gene is testis-specific and occurs during the early stages of germ cell differentiation. In a comparative study, the location of the hSMP-I protein in sperm and in germ cells of the seminiferous tubules of rats was determined. The target antigen was immunolocated on the head and tail of rat sperm and in late spermatids and spermatozoa of rat testis. These results suggest that, in the rat, the HSD-1 gene is expressed during spermiogenesis.

Identification of a rabbit epididymal protein gene.

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.