Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Identification of human sperm peptide sequence involved in egg binding for immunocontraception.

Development of a vaccine based on sperm antigens represents a promising approach to contraception. The sperm-zona pellucida (ZP) interaction constitutes the most important event in the fertilization process, and the molecular sequences involved at this site may provide the most attractive candidates for immunocontraception. In the present study, using the phase peptide display technique, a novel dodecamer sequence, designated as YLP(12), was identified that is involved in sperm-ZP recognition/binding. The synthetic 12-mer peptide based on this sequence and its monovalent Fab' antibodies specifically and significantly (P < 0.05) inhibited human sperm-ZP binding. In Western blot and immunoprecipitation procedures, the YLP(12) peptide recognized the ZP3 component of solubilized human ZP proteins. In the Western blot procedure involving 10 different human tissue extracts, the anti-YLP(12) Fab' antibodies recognized a protein band of approximately 72 +/- 2 kDa only in the testis lane. The peptide sequence was localized on the acrosomal region of the human sperm cell. These findings indicate that the novel testis-specific 12-mer YLP(12) that is present in the acrosomal region and is involved in human sperm-ZP interaction may find applications in contraceptive vaccine development, as well as in diagnosis and treatment of male infertility mediated through sperm dysfunction.

Fertilization antigen (FA-1) completely blocks human sperm binding to human zona pellucida: FA-1 antigen may be a sperm receptor for zona pellucida in humans.

The effects of purified human sperm fertilization antigen-1 (FA-1), affinity-purified monoclonal Fab' antibody to FA-1, and monoclonal Fab' antibody to phosphotyrosine residues on human sperm-zona interaction were investigated. The purified FA-1 antigen completely blocked sperm binding to zona pellucida (P < 0.0001). Also, the monoclonal Fab' antibodies to FA-1 antigen and phosphotyrosine residues significantly (P < 0.05) reduced sperm-zona pellucidae and the antibodies were preincubated with sperm before insemination and not vice versa. These results suggest that the tyrosine phosphorylation especially of FA-1 antigen has an important role in zona pellucida receptor recognition and binding. These findings also suggest that FA-1 antigen may be the sperm receptor involved in zona pellucida binding in humans.

Human testis vitamin D binding protein involved in infertility.

Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman containing antisperm antibodies that induced head-to-head agglutination of human sperm. The antibodies in the IgG fraction interacted with a 60-kD protein found in human testes determined by Western blot. The 60-kD protein was purified from human testis by isoelectric focusing (IEF), affinity chromatography on blue sepharose column, and preparative electrophoresis with electroelution. The purified 60-kD protein migrated as a single homogeneous band when analyzed by SDS-PAGE. The amino acid sequence of the N-terminus was determined. The initial 10 amino acid residues were identical to the human serum vitamin D binding protein (VDBP). Polyclonal antibodies were raised against the 60-kD protein. The polyclonal anti-60-kD antibodies and the anti-VDBP antibodies obtained from a commercial source immobilized human sperm in vitro. The interacting antigens were located on the postacrosomal region and midpiece of human sperm, as determined by an immunofluorescence method. The IgG fraction prepared from the serum of an infertile woman interacted with the human testis 60-kD protein but failed to stain serum VDBP. The results suggest that the 60-kD and VDBP are related proteins but not identical entities and that the 60-kD protein contains a unique structural group lacking in serum VDBP. Production of antibodies against the unique structure of the 60-kD protein may be the cause of the infertility.

Identification of human seminal plasma components that bind IgG2 variant.

Human seminal plasma (hsp) contains soluble proteins capable of binding immunoglobulin (Ig) G. Two novel components with estimated molecular sizes of 90 and 21 kD interact specifically with a variant of IgG2 found in 20% of human sera tested. The common IgG2 present in human sera and other subclasses of IgG did not bind with the hsp components. The present findings shows that the interacting IgG2 is a variant and not the common or prevalent species. The 90-kD component of hsp with IgG2 binding property is probably a nonglycosylated protein, whereas the 21-kD component is a glycosylated protein. The 90- and 21-kD components were detected in 20% of hsp specimens tested. Thus they are not present in the majority of hsp. Since the IgG2 binding components of hsp and the serum IgG2 variant are found in 20% of men and 20% of individuals, respectively, they can be used as genetic markers.

Identification of a cAMP-dependent protein kinase in bovine and human follicular fluids.

A soluble protein kinase (PK) was purified from bovine and human follicular fluids (FF) by ultrafiltration through a PM-10 membrane followed by chromatography on heparin-agarose, DEAE-cellulose and cellulose phosphate columns. The PK phosphorylated calf thymus histones and endogenous FF proteins having estimated Mrs of 40, 62, 128 and 180 KD. cAMP enhanced PK activity; whereas protein kinase A (PKA)-inhibitor peptide blocked the activity. The present findings suggest that the enzyme is a cAMP-dependent PK.

Inhibition of progesterone action by a factor in human follicular fluid.

A factor was found in human follicular fluid that blocked progesterone-stimulated net uptake of 45Ca2+ in human sperm and progesterone-induced maturation of Xenopus oocyte. The factor was partially purified by ultrafiltration through PM-10 membrane and gel filtration on Sephadex G-25 column. The active fraction is effective at a concentration of 200 micrograms/ml. The present findings suggest that hFF factor may regulate the metabolism of progesterone sensitive cells.

A follicular fluid factor inhibiting Xenopus oocyte maturation.

A factor was isolated from bovine follicular fluid (FF) that blocked progesterone-induced maturation of Xenopus oocytes. The factor was purified by ultrafiltration through PM-10 membrane, gel filtration on Sephadex G-25 column, gel permeation chromatography on Superose 12 column and reversed-phase HPLC on Partisil ODS-3 column. The active factor is a peptide with an estimated Mr of 8000 and it effectively blocked progesterone-induced maturation of Xenopus oocytes at a concentration of 100 micrograms/ml. On the other hand, it did not influence the spontaneous maturation of isolated cumulus-enclosed or denuded mouse oocytes. The present findings suggest that the FF factor may be an inhibitor or antagonist of progesterone action.

Identification of hypoxanthine in bovine follicular fluid.

Bovine follicular fluid (bFF) blocks the occurrence of spontaneous germinal vesicle breakdown (GVBD) of isolated mouse oocytes. One of the active factors was purified by ultrafiltration through a PM-10 membrane filter, gel filtration through Sephadex G-25, and HPLC using a reversed-phase ODS-3 column and a Superose 12 column. This factor was identified as hypoxanthine by gas chromatography and mass spectrometry. The purified substance and hypoxanthine showed the same retention time on HPLC, and identical UV absorbance and mass spectra. There are other oocyte maturation inhibitors in follicular fluid that should be identified.

Shortening of luteal phase in common marmosets by sheep ovarian follicular fluid peptide.

A low molecular weight peptide has been partially purified from sheep follicular fluid. It inhibited FSH binding to granulosa cells from ovarian follicles of common marmosets (Callithrix jacchus). When injected into cycling marmosets during the follicular phase, it reduced the area under the curve (AUC) of circulating progesterone. The peptide also shortened the luteal phase in all marmosets during the treatment cycle compared to the pretreatment control cycle. These results indicate that the ovarian follicular fluid peptide inhibited FSH binding to granulosa cells thereby probably resulting in decreased progesterone secretion (AUC) from these cells and subsequently inducing luteal insufficiency.

Control of follicular maturation in the mouse by a nonsteroidal regulator from sheep follicular fluid.

Follicular fluid from sheep ovaries was gel-chromatographed on Sephadex G-100, and the retarded active fraction (Fr-IV, Kav = 1) was rechromatographed on G-25 (GF2). When injected into pseudopregnant mice, it did not affect corpus luteum function, but at the time of the termination of pseudopregnancy ovulation was suppressed. A dose of 20 micrograms GF2 when injected into cycling mice inhibited follicular maturation and ovulation. Plasma progesterone levels were also reduced, indicating the role of a nonsteroidal factor in follicular maturation, ovulation, and luteinization.

Inhibition of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the granulosa cells of mouse ovarian follicles by follicular fluid peptide.

A protein fraction (GF2) was purified from sheep ovarian follicular fluid. Its action on 3 beta-HSD activity in the mouse granulosa cells was measured in an in vitro system. The fraction (GF2) caused dose-dependent depletion of the 3 beta-HSD activity in granulosa cells and progesterone in the spent medium. A maximum inhibition of the activity was achieved after 30 min incubation of the granulosa cells with the GF2 fraction. Further purified HPLC fraction (Fr1) also inhibited 3 beta-HSD activity. In vivo administration of the GF2 fraction in normal cycling female mice also decreased the 3 beta-HSD activity in the granulosa cells of the ovarian follicles and plasma progesterone levels indicating the GF2 fraction to be a 3 beta-HSD inhibitor.