Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

No abstract

Serum anti-ovarian antibodies (AOAs) have been shown in autoimmune premature ovarian failure and in vitro fertilization-embryo transfer (IVF-ET) cases. The specificity of assays detecting these antibodies has been questioned. Researchers have used several techniques (e.g., ELISA and indirect immunofluorescence). Few have reported on the non-specificity and the type of molecular and cellular targets. We reported earlier on the presence of naturally occurring anti-albumin antibodies as the likely factor for non-specificity. Having developed a novel blocking recipe, we show substantial elimination of this non-specificity. With these standardized tests, we hereby report multiple targets at protein and histological levels. In our study group, 15 of 50 (30%) patients with premature ovarian failure and 13 of 50 (26%) IVF-ET patients showed the presence of AOAs. Western blotting showed a large number of patients making AOAs to a 90-kDa protein, followed by 97- and 120-kDa proteins. Histochemically, it was evident that the sera of these patients predominantly react with the oocyte; other somatic cellular targets are also involved. The specific non-invasive test developed by us was found to be useful because it could carry out a reliable diagnosis of an autoimmune etiology that would be very helpful to select patients in whom immune-modulating therapy could be recommended, which in turn may restore ovarian function and fertility.

Autoimmunity is a well-established causative factor of premature ovarian failure (POF), and evidence for the same has been well reported in the literature. Detection of specific autoantibodies remains the most practical clinical research marker of any autoimmune disease. Variation in efficiency and specificity in the detection of ovarian autoantibodies has been reported. However, the frequency of false positivity and a solution to overcome this has not yet been reported. Herein, we report autoantibody to albumin as the likely responsible agent for false positivity. Our data indicate that presence of naturally existing autoalbumin antibodies in the circulation of normal women is responsible for the false signal seen in SDS-PAGE Western blot analysis and in immunohistochemistry (IHC). Having shown the presence of anti-albumin antibody in normal women as well as in the sera of POF patients, we have developed a novel blocking agent to overcome this problem. A high titer polyclonal antibody against human serum albumin was generated. This antibody showed immunoreactivity to albumin obtained from various sources. Preincubation of Western blots and IHC sections with this antibody drastically reduced background signals. The advantage of using this blocking was evident by identification of specific anti-ovarian antibodies in a group of POF patients. This blocking procedure made it possible to obtain a clear indication of the ovarian antibody status in women presenting with autoimmune POF.