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.

Student perceptions of an upper-level, undergraduate human anatomy laboratory course without cadavers.

Several programs in health professional education require or are considering requiring upper-level human anatomy as prerequisite for their applicants. Undergraduate students are confronted with few institutions offering such a course, in part because of the expense and logistical issues associated with a cadaver-based human anatomy course. This study describes the development of and student reactions to an upper-level human anatomy laboratory course for undergraduate students that used a regional approach and contemporary, alternative teaching methods to a cadaver-based course. The alternative pedagogy to deliver the curriculum included use of commercially available, three-dimensional anatomical virtual dissection software, anatomical models coupled with a learning management system to offer Web-based learning, and a new laboratory manual with collaborative exercises designed to develop the student's anatomical skills and collaborative team skills. A Likert-scale survey with open-ended questions was used to ascertain student perceptions of the course and its various aspects. Students perceived that the noncadaver-based, upper-level human anatomy course with an engaging, regional approach is highly valuable in their learning of anatomy. anatomy.

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.

The cancer screening project for women: experiences of women who partner with women and women who partner with men.

The Cancer Screening Project for Women is a study about the experiences of legally unmarried women with breast, cervical, and colorectal cancer screening. During the initial phase of the study, we conducted focus groups to explore factors that influence unmarried women's decisions about cancer screenings. Women were invited to attend one of four group discussions: (1) never married women who either partner with women (WPW) or with both women and men (WPWM), (2) previously married women who now partner either with women (WPW) or with both women and men (WPWM), (3) never married women who partner with men (WPM), and (4) previously married women who partner with men (WPM). Twenty-eight women attended the focus groups, 14 WPW and 14 WPM. Several barriers to screening were consistent across the groups and included lack of acknowledgement and validation in medical settings, administrative barriers, pain, and concerns about body image. WPW specifically discussed fears about discrimination if and when they acknowledge their sexual orientation. WPW also described how women who express their gender androgynously are more likely to avoid health care facilities. Further studies are needed to determine if the themes we identified are consistent among larger samples of unmarried women.

DNA topoisomerase II distribution in mouse preimplantation embryos.

DNA topoisomerase II (topo II) is an essential enzyme that mediates a variety of chromosome activities including DNA replication, transcription, recombination, and chromosome condensation and segregation. Isoform-specific anti-topo II antibodies were used to determine the distribution of topo II alpha and beta in mouse gametes and embryos. Immunoblot analysis with two anti-topo IIalpha antibodies revealed that a 170 kDa topo IIalpha band was present in ovary and testis. Mature sperm exhibited an 89 kDa band only, which may be a degradation product of topo IIalpha. Immunoblots probed with a monoclonal antibody that recognizes both isoforms, showed bands at 170 and 180 kDa, which correspond to topo IIalpha and beta, respectively. An additional 100 kDa band was also present in ovary and testis. Mature sperm did not exhibit staining with this antibody. We also localized topo II in mouse gametes and embryos up to the blastocyst stage using immunofluorescence microscopy. While both isoforms were found in nuclei and nucleoli of germinal vesicle oocytes, topo IIalpha localized to metaphase chromosomes during meiosis, and only to nucleoli during embryonic interphase. Topo IIbeta was absent from chromosomes of metaphase II oocytes, but localized to embryonic interphase nuclei. Both full-length isoforms were absent from sperm, indicating topo II is stored maternally. These results identify topo II as an important component of mouse oocyte and embryonic chromatin, and suggest its involvement in oocyte maturation and preimplantation embryonic development. The different immunofluorescent staining patterns indicate topo IIalpha and beta may serve different roles during the embryonic cell cycle.

DNA topoisomerase II is essential for preimplantation mouse development.

Topoisomerase II (topo II) is an essential enzyme that alters DNA topology. This activity is important for a variety of chromosome functions including DNA replication, transcription, recombination, and chromosome condensation and segregation. Previously we localized topo II in mouse gametes and preimplantation embryos using isoform-specific antibodies demonstrating the presence of the enzyme in oocytes and embryos, but not sperm. To probe functions of topo II during preimplantation development, we treated mouse zygotes with 100 nM teniposide, and assessed embryo morphology and DNA replication. Teniposide blocked cleavage in 69% embryos; the remainder cleaved once but had abnormal nuclei. Teniposide-treated embryos were devoid of topo II immunofluorescence. Teniposide also prevented DNA replication, implicating topo II in this process. Embryos treated with a 2 hr pulse of teniposide recovered and developed to the blastocyst stage, indicating 100 nM teniposide did not induce apoptosis. To more specifically analyze topo IIalpha function, we treated zygotes with topo IIalpha-targeted antisense oligodeoxynucleotides. Most zygotes arrested at the 2-cell stage while controls developed into blastocysts indicating topo IIalpha is essential for preimplantation development. The absence of topo IIalpha, but not beta immunofluorescence in antisense-treated embryos confirms the specificity and impact of the treatment. In addition, topo IIalpha is newly synthesized at the 2-cell stage. These results establish an essential function for topo II in mouse preimplantation embryonic development.