A unique lineage gives rise to the meibomian gland.

PURPOSE: To identify the lineage that contributes to the morphogenesis of the meibomian gland. METHODS: To examine which cell lineage gives rise to the meibomian gland, the expression of Pax6 as well as that of various cytokeratin markers, including keratin 14 (Krt14), Krt15, Krt4, and Krt10, was examined with immunofluorescent staining of C57BL/6J mouse eyelids from P2 to P11 pups and adult mice. RESULTS: Pax6 was localized to the cytoplasm within the acinar region of the meibomian glands during morphogenesis but was absent in the fully developed gland. Keratin 14 was expressed throughout the gland at all stages whereas keratin 15 was absent at all stages. Keratin 4, a marker of mucosal lineage, was present throughout the gland and was colocalized with keratin 10 (epidermal lineage marker) in the developing duct at P4. This colocalization region decreased as the gland developed becoming restricted to the central duct near the opening to the acini in the fully developed gland. CONCLUSIONS: We identified a unique cell lineage that expresses markers characteristic of mucosal and epidermal epithelia during meibomian gland morphogenesis. This unique group of cells was located in the central duct with a concentration near the ductule orifice. The expression of these cells reduced during meibomian gland morphogenesis and may play a role in the development and homeostasis of the gland.

Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality.

The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction.

Imaging the zona pellucida of canine and feline oocytes using scanning electron microscopy.

Ultrastructure of the zona pellucida (ZP) of canine and feline oocytes has not been fully investigated. The objective of the study was to evaluate the potential use of the low vacuum scanning electron microscope (LVSEM) with oocytes. This required development of a method to prepare canine and feline cumulus-oocyte complexes (COCs) for LVSEM to provide ultrastructural information on the ZP. COCs were collected from ovaries, and cumulus cells were either partially or completely removed to reveal the ZP. COCs and zona-intact oocytes were fixed at 4 degrees C for 1 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and subsequently viewed wet or further processed by critical point drying, and viewed uncoated or sputter coated with gold. Although the spongy surface of the ZP was visible at low vacuum in uncoated oocytes, coated oocytes had more details at high vacuum. The ZP surface of canine and feline oocytes contained numerous various-sized, spherical or elliptical pores that narrowed centripetally splitting into several smaller, deep pores. The round to oblong cumulus cells tightly surrounded the ZP. Each corona radiata cumulus cell tapered into a thin projection that entered the ZP. Our detailed techniques will enable future studies connecting ultrastructural and molecular aspects of oocyte maturation and development in mammals.