Autoantibodies: Key Mediators of Autoimmune Infertility

Autoimmune diseases have gender bias with predominance in females, autoimmune infertility (AI) being no exception. This chapter will focus on AI in females with brief reference to the same in males. Autoimmune diseases have established protocols for detection and management of ensuing infertility, however similar protocols for unexplained infertility [tubal blockage, endometriosis, premature ovarian insufficiency (POI), undiagnosed underlying autoimmune disease (Sjögren’s syndrome, IBS, celiac disease) and tubal blockage] are not established. Endometriosis and POI, in particular, have autoimmune etiology yet lack specific and sensitive biomarkers for accurate diagnosis. If autoantibodies are indeed diagnosed, then treatment regimen focuses on AI which has known adverse effects. The detection of natural antibodies as autoantibodies presents a viable alternative to organ specific biomarker panel for better management of AI

Fertilization, embryonic development and oviductal environment: Role of estrogen induced oviductal glycoprotein

1043-1055Mammalian oviduct is the physiological site for sperm capacitation, gamete fertilization and early embryonic development. The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) in response to estrogen. The protein has been shown to interact with gametes and early embryo. Several key functions have been postulated particularly its role in pre-implantation events which would have far reaching implications in assisted reproductive technology and in the development of non-hormonal contraceptive vaccine. The intention of this article is to discuss the current status of the protein and analyze how far the postulated function of OGP has been borne out by the available data

Cystic fibrosis transmembrane conductance regulator (CFTR) gene abnormalities in Indian males with congenital bilateral absence of vas deferens & renal anomalies

Background & objectives: The role of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in congenital bilateral absence of vas deferens and unilateral renal agenesis (CBAVD-URA) has been controversial. Here, we report the cases of five Indian males with CBAVD-URA. The objective was to evaluate the presence or absence of CFTR gene mutations and variants in CBAVD-URA. The female partners of these males were also screened for cystic fibrosis (CF) carrier status. Methods: Direct DNA sequencing of CFTR gene was carried out in five Indian infertile males having CBAVD-URA. Female partners (n=5) and healthy controls (n=32) were also screened. Results: Three potential regulatory CFTR gene variants (c.1540A>G, c.2694T>G and c.4521G>A) were detected along with IVS8-5T mutation in three infertile males with CBAVD-URA. Five novel CFTR gene variants (c.621+91A>G, c.2752+106A>T, c.2751+85_88delTA, c.3120+529InsC and c.4375-69C>T), four potential regulatory CFTR gene variants (M470V, T854T, P1290P, Q1463Q) and seven previously reported CFTR gene variants (c.196+12T>C, c.875+40A>G, c.3041-71G>C, c.3271+42A>T, c.3272-93T>C, c.3500-140A>C and c.3601-65C>A) were detected in infertile men having CBAVD and renal anomalies Interpretation & conclusions: Based on our findings, we speculate that CBAVD-URA may also be attributed to CFTR gene mutations and can be considered as CFTR-related disorder (CFTR-RD). The CFTR gene mutation screening may be offered to CBAVD-URA men and their female partners undergoing ICSI. Further studies need to be done in a large sample to confirm the findings

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

<div><p>Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.</p> </div

Co-immunoprecipitation of PSP94 and PAP proteins from fraction III.

<p><b>A.</b> PSP94-PAP complex from fraction III (50 µg) co-immunoprecipitated with anti-PAP antibody showing the presence of PSP94 (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with mouse isotype control antibody (lane 1) served as controls. 10 µg of fraction III was loaded in lane 4 as input and the immunoblot was probed with anti-PSP94 antibody. <b>B.</b> PSP94-PAP complex from fraction III (50 µg) was co-immunoprecipitated with anti-PSP94 antibody showing the presence of PAP (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with normal rabbit serum (lane 1) served as controls. 10 µg of fraction III was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. Molecular weight markers shown are in kDa.</p

Mass spectrometric analysis of spots excised from the 2D gel.

*<p>Note: Discrepancy in the isoelectric point (pI) or molecular weight (MW) of the proteins detected here with that of the protein entries in NCBI database could be probably due to the post-translational modifications or protein processing.</p

Two-dimensional gel electrophoresis profile of fraction III.

<p>The 2D gel resolved by isoelectricfocusing in the first dimension (pH range 3–10) followed by SDS-PAGE in the second dimension was stained with silver nitrate. The protein spots (circled) were excised and subjected to MS and MS/MS analysis (Data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058631#pone-0058631-t001" target="_blank">Table 1</a>). Molecular weight markers shown are in kDa.</p

Identification of proteins in fraction III.

<p><b>A.</b> MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. <b>B.</b> Immunoblot analysis of fraction III probed with anti-PAP antibody. Molecular weight markers shown are in kDa.</p

Interaction of pure PSP94 and PAP proteins in vitro.

<p>500 ng of PSP94 incubated with or without PAP (500 ng) (lane 1 and 2 respectively) and immunoprecipitated using anti-PAP antibody. Pure PSP94 (20 ng; lane 3) and PAP (500 ng; lane 4) proteins were loaded as input. Lanes 1, 2 and 3 were immunoblotted with anti-PSP94 antibody, while lane 4 was immunoblotted with anti-PAP antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.</p

Fractionation of human seminal plasma proteins.

<p><b>A.</b> Preparative RP-HPLC profile of seminal plasma proteins (bound fraction from Phenyl Sepharose chromatography) showing three major peaks (fractions I, II and III). <b>B.</b> Immunoblot of fractions I, II and III probed with anti-PSP94 antibody. <b>C.</b> One dimensional SDS-PAGE profile of fractions I, II and III. Molecular weight markers shown are in kDa.</p