Abstract

In a previous study we found that the tear film of pinnipeds lacks a lipid layer, leading us to hypothesize that mucin-type glycoproteins are the major protective components at the ocular surface of marine mammals. The purpose of this study was to characterize mucin-type O-glycans in marine mammal tears, and to determine whether pinnipeds have meibomian glands. Protein and carbohydrate concentrations of tears collected (with IRB & AICUC approvals) from humans, pinnipeds, and cetaceans were determined using bicinchoninic acid and sulfuric methods respectively. We found that marine mammal tears have a much greater ratio of carbohydrate to protein as compared to that of human tears. Samples normalized for protein were electrophoresed on 1% SDS-agarose gels, transferred to nitrocellulose membranes by vacuum blotting, and probed for the T-antigen, which is specific to mucin carbohydrates, using Arachis hypogea agglutinin (PNA), followed by chemiluminescence detection. There was PNA binding to proteins of high molecular weight, consistent with mucins, in human, sea lion, and dolphin tear samples. The size distribution of PNA-positive bands was different for each species analyzed, with dolphin migrating at the highest molecular weight. Normal-phase high-pressure liquid chromatography (HPLC) was used to analyze O-glycans released from tear samples using ammonia-based beta-elimination. Separation of O-glycans by HPLC revealed differences in chromatograms of tears from humans, sea lions, and dolphins. For histology, ocular tissues from a sea lion necropsy were fixed in formalin, examined using a stereo magnifier, then embedded in paraffin, sectioned to 10–20 µm thickness using a rotary microtome, and stained. In stereo magnifier analysis, a distinct tarsus was missing. The lid consisted mainly of muscular tissue, and underneath the epidermis there was a whitish granular layer consisting of hair follicles, with bundles of sebaceous acini between hair follicles. Towards the lid margin, the number of hair follicles decreased and the relative volume of sebaceous glands increased, forming several solitary glandular bodies at the margin. The acini drained via ductules into a straight duct with a multi-layered stratified squamous epithelium, which opened onto the outer lid skin. Our results show that mucin-type O-glycans are present in marine mammal tears, as has been shown in humans; however, there are differences in size distribution and O-glycan profiles across species. Pinnipeds have sebaceous glands at the eyelid margin that resemble human meibomian glands in structure, but are different in orientation and size.

Acknowledgements

We thank the staff and trainers at Dolphin Quest Oahu, Oahu, HI; Aquarium of Niagara, Niagara Falls, NY; and New England Aquarium, Boston, MA, for their very generous support in collecting marine mammal tear samples for this project. We also thank the Marine Mammal Center, Sausalito, CA for their generous contributions of ocular tissues.