Eusebio S. Pires

Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization

Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.

Multiplicity of molecular and cellular targets in human ovarian autoimmunity: an update

PurposeTo provide an update of putative auto-antigens identified and proposed to be involved in human ovarian autoimmunity.MethodsReview of literature pertaining to ovarian auto-antigens / proteins identified with various immunological tools using sera of infertile women as a probe for investigation.ResultsAn overview of autoimmune targets known till date in the study of human ovarian autoimmunity.ConclusionsAnti-ovarian antibodies (AOA) to multiple components and compartments of the ovary are present in the sera of infertile women. Researchers propose that these AOA may be responsible for ovarian failures and therefore render women to be infertile. Evaluation of AOA can be effective as a prognostic factor in the treatment of infertile patients and for the IVF-ET program.

SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition

Background: Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra‐acrosomal sperm protein SLLP1. Results: Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary‐secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti‐podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. Conclusions: The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies. Developmental Dynamics, 242:1405–1426, 2013.

Can anti-ovarian antibody testing be useful in an IVF-ET clinic?

ObjectiveTo establish importance of anti-ovarian antibodies (AOA) testing in infertile women.DesignA clinical reproductive outcome comparative study between two groups of women undergoing IVF-ET. Group 1 consists of women tested positive for AOA, put on corticosteroid therapy, reverted to AOA negative and then taken up for IVF-ET. Group 2 were seronegative for AOA.SettingMajor urban infertility reference centre and National research institute.Patient(s)Five hundred seventy infertile women enrolled for IVF-ET.Intervention(s)AOA testing, corticosteroid therapy and IVF-ET/ICSI.Main outcome measure(s)Comparable clinical outcome and significance of AOA testing established.ResultsAOA positive serum samples were sent periodically to re-investigate presence of AOA after corticosteroid therapy and women turned AOA negative were taken up for IVF-ET. Of the 70/138 women in group 1 who were treated with corticosteroids and turned seronegative for AOA, 22/70 were poor responders and needed donor oocyte-recipient cycles. Results demonstrated that fertilization and clinical pregnancy rates between both groups are comparable. Nevertheless, it is also observed that there is poor response to stimulation protocol, smaller number of oocytes retrieved and more spontaneous abortions in group 1 women. Hence not all outcomes following the treatment are comparable between the two groups. Usefulness of the test was established in two case studies.ConclusionsAOA testing could be included in the battery of tests investigating and treating infertility.

Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors

The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.

The Unmysterious Roles of HSP90: Ovarian Pathology and Autoantibodies

The heat shock proteins (HSPs) are a group of evolutionarily conserved proteins with important physiological functions, whose synthesis is enhanced by elevated temperature or other stresses. HSPs show high sequence homology between different species, from bacteria to humans. Despite the significant degree of evolutionary conservation, HSPs are highly immunogenic. Of the several HSPs, HSP90 is an abundant, constitutively expressed chaperone constituting around 1-2% of total cellular protein under non-stress conditions. This protein from even the most distantly related eukaryotes has 50% amino acid identity, and all have more than 40% identity with the Escherichia coli protein. They are immunodominant antigens for many common microbes, and thus their epitopes are recognized by the immune system. As HSPs are overexpressed at sites of acute and chronic inflammation, individuals are likely to be sensitized during the course of a microbial infection encountered during life. This chapter considers the evidence of a role for HSP90 in autoimmune ovarian failure, where autoantibodies to it have been observed in patients, and has been correlated to infertility.

Heat Shock Protein 90: Truly Moonlighting!

Hsp90 is an essential and abundantly expressed molecular chaperone in any living cell. The multiplicity of Hsp90 cellular functions is driven by its interaction with a broad range of partner proteins and thereby establishing itself as a moonlighting molecule. There are newer insights emerging to ascertain the cellular and physiological roles of Hsp90, such as (and not limited to) chromatin remodeling, gene regulation and developmental pathways. Hsp90 has been recognized as an important therapeutic target and has been linked to an increasing number of diseases, including cancer. Development of Hsp90 therapeutic reagents would be valuable research tools towards the maintenance of the proteome in health and disease. This review revisits the expression, structure-function, and clinical significance of the Hsp90 and its forms and reinforces its impact as a disease target.