Lynae M. Brayboy

The biology of the germ line in echinoderms.

The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ‐line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ‐line determination in echinoderms, an early‐branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ‐line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ‐line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser‐known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ‐line fate determination. This enables a strongly contrasted picture for germ‐line determination in this phylum, but one for which transitions between different modes of germ‐line determination might now be experimentally addressed. Mol. Reprod. Dev. 81: 679–711, 2014.

Simple perfusion apparatus for manipulation, tracking, and study of oocytes and embryos

OBJECTIVE To develop and implement a device and protocol for oocyte analysis at a single cell level. The device must be capable of high resolution imaging, temperature control, perfusion of media, drugs, sperm, and immunolabeling reagents all at defined flow rates. Each oocyte and resultant embryo must remain spatially separated and defined. DESIGN Experimental laboratory study. SETTING University and academic center for reproductive medicine. PATIENT(S)/ANIMAL(S) Women with eggs retrieved for intracytoplasmic sperm injection (ICSI) cycles, adult female FVBN and B6C3F1 mouse strains, sea stars. INTERVENTION(S) Real-time, longitudinal imaging of oocytes after fluorescent labeling, insemination, and viability tests. MAIN OUTCOME MEASURE(S) Cell and embryo viability, immunolabeling efficiency, live cell endocytosis quantification, precise metrics of fertilization, and embryonic development. RESULT(S) Single oocytes were longitudinally imaged after significant changes in media, markers, endocytosis quantification, and development, all with supreme control by microfluidics. Cells remained viable, enclosed, and separate for precision measurements, repeatability, and imaging. CONCLUSION(S) We engineered a simple device to load, visualize, experiment, and effectively record individual oocytes and embryos without loss of cells. Prolonged incubation capabilities provide longitudinal studies without need for transfer and potential loss of cells. This simple perfusion apparatus provides for careful, precise, and flexible handling of precious samples facilitating clinical IVF approaches.

Multidrug-resistant transport activity protects oocytes from chemotherapeutic agents and changes during oocyte maturation

OBJECTIVE To determine the multidrug-resistant transporter (MDR) activity in oocytes and their potential role in oocyte susceptibility to chemotherapy. DESIGN Experimental laboratory study. SETTING University and academic center for reproductive medicine. SUBJECT(S) Women with eggs retrieved for intracytoplasmic sperm injection cycles and adult female FVBN and B6C3F1 mouse strains. INTERVENTION(S) Inhibition of MDR activity in oocytes. MAIN OUTCOME MEASURE(S) Efflux activity of MDRs with the use of quantitative fluorescent dye efflux, and oocyte cell death when exposed to chemotherapy. RESULT(S) Oocytes effluxed fluorescent reporters, and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. Geminal vesicle oocytes were more efficient at efflux than metaphase 2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death with the use of two different viability assays compared with control samples and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes, with the greatest accumulation in the geminal vesicle stage. CONCLUSION(S) Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis and that these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up-regulation of these transporters during gonadotoxic therapy.

Multidrug resistance transporter-1 and breast cancer resistance protein protect against ovarian toxicity, and are essential in ovarian physiology

Ovarian protection from chemotoxicity is essential for reproductive health. Our objective is to determine the role of ATP-dependent, Multidrug Resistance Transporters (MDRs) in this protection. Previously we identified MDR-dependent cytoprotection from cyclophosphamide in mouse and human oocytes by use of MDR inhibitors. Here we use genetic deletions in MDR1a/b/BCRP of mice to test MDR function in ovarian somatic cells and find that mdr1a/b/bcrp-/- mice had significantly increased sensitivity to cyclophosphamide. Further, estrus cyclicity and follicle distribution in mdr1a/b/bcrp-/- mice also differed from age-matched wildtype ovaries. We found that MDR gene activity cycles through estrus and that MDR-1b cyclicity correlated with 17β-estradiol surges. We also examined the metabolite composition of the ovary and learned that the mdr1a/b/bcrp-/- mice have increased accumulation of metabolites indicative of oxidative stress and inflammation. We conclude that MDRs are essential to ovarian protection from chemotoxicity and may have an important physiological role in the ovary.

The double-edged sword of the mammalian oocyte – advantages, drawbacks and approaches for basic and clinical single cell analysis

Oocytes are usually the largest cells in the body and as such offer unique opportunities for single-cell analysis. Unfortunately, these cells are also some of the rarest in the mammalian female, usually necessitating single-cell analysis. In cases of infertility in humans, determining the quality of the oocyte is often restricted to a morphological analysis or to the study of cellular behaviors in the developing embryo. Minimally invasive approaches could greatly assist the clinician to prioritize oocytes for fertilization or following fertilization, which embryo to transfer back into the woman. Transcriptomics of human and mouse oocytes may have great utility, and recently it was learned that the polar body faithfully reflects the transcript prevalence in the oocyte. The polar body may thus serve as a minimally invasive proxy for an oocyte in the clinic. In the mouse, the transcriptomes of oocytes from mice of the same strain are markedly similar; no significant differences are apparent in transcript prevalence or identity. In human oocytes however, the transcript pool is highly variable. This is likely the result of different histories of each oocyte, in the age of the donor woman, the different hormonal exposures and the prolonged time from specification of the primary oocyte to the fully grown and ovulated egg. This variability in human oocytes also emphasizes the need for cell-by-cell analysis of the oocytes in vitro; which oocytes have a better potential for fertilization and development? To this end, new imaging capabilities are being employed. For example, a single-cell analytical device for oocytes (the simple perfusion apparatus, or SPA) enables investigators to load multiple oocytes in individual wells, to visualize them on the microscope and to use controlled temperature and media flow by perfusion for optimal clinical applications. Recently, developed Raman microspectroscopy approaches suggest that this imaging modality may enable more in-depth analysis of the molecular characteristics of an oocyte that, in combination with the SPA and transcriptomic approaches, might assist the clinician to prioritize more effectively human oocytes and embryos for transfer into women. This review is intended to update the reader on the status of the examination of single oocytes from a variety of approaches and to emphasize areas that may be primed for advancement in the near future.

Nitrogen mustard exposure perturbs oocyte mitochondrial physiology and alters reproductive outcomes

Nitrogen mustard (NM) is an alkylating chemical warfare agent, and its derivatives are used in chemotherapy. Alkylating agents can cause mitochondrial damage, so exposed females may transmit damaged genomes to their children, since mitochondria are maternally inherited and oocytes are not thought to undergo mitophagy (Boudoures et al. [1]). The objective of this study is to investigate NMs effects on oocyte mitochondria to understand risks facing female soldiers, cancer patients, and their children. Mice were injected intraperitoneally with NM, monitored for reproductive outcomes, and ovaries and oocytes were isolated for analysis. Escalating doses of NM increased oxidative stress in parental and F1 generation oocytes, suggesting that mitochondrial damage by NM is enhanced by mitochondrial superoxide. NM-treated ovaries in vitro exhibited smaller mitochondrial volume, more electron-dense and multivesicular structures, and lower birth weight litters. These results demonstrate that females must be protected from alkylating agents for their health, and the health of their offspring.