Amy Inselman

A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse.

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.

Temporal expression of cell cycle-related proteins during spermatogenesis: establishing a timeline for onset of the meiotic divisions

During spermatogenesis, the complex events of the first meiotic prophase and division phase bring about dramatic changes in nuclear organization. One factor frustrating mechanistic dissection of these events is lack of knowledge about precisely what events occur, in what order they occur, and how they may be interrelated by temporal sequence; in other words, a precise timeline is lacking. This temporal ordering problem can be tackled by following expression and localization in mouse spermatocytes of proteins critical to events of the meiotic cell division process. These include ones that are primarily chromosomal and related to pairing and recombination, as well as kinases and substrates that mediate the cell cycle transition. Distinct and protein-specific patterns occur with respect to expression and localization throughout meiotic prophase and division and dramatic relocalization of proteins occurs as spermatocytes enter the meiotic division phase. This information provides a foundation for a meiotic timeline that can be augmented to provide, eventually, a complete catalog of meiotic events and their temporal sequence. Such a framework can clarify mechanisms of normal meiosis as well as mutant phenotypes and aberrations of the meiotic process that lead to aneuploidy.

Mitogen-Activated Protein Kinase Dynamics During the Meiotic G2/MI Transition of Mouse Spermatocytes

Abstract Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.

Alternative models in developmental toxicology

In light of various pressures, toxicologists have been searching for alternative methods for safety testing of chemicals. According to a recent policy in the European Union (Regulation, Evaluation Authorisation and Restriction of Chemicals, REACH), it has been estimated that over the next twelve to fifteen years, approximately 30,000 chemicals may need to be tested for safety, and under current guidelines such testing would require the use of approximately 7.2 million laboratory animals []. It has also been estimated that over 80%% of all animals used for safety testing under REACH legislation would be used for examining reproductive and developmental toxicity [Hofer et al., 2004]. In addition to REACH initiatives, it has been estimated that out of 5,000 to 10,000 new drug entities that a pharmaceutical company may start with, only one is finally approved by the Food and Drug Administration at a cost of over one billion dollars []. A large portion of this cost is due to animal testing. Therefore, both the pharmaceutical and chemical industries are interested in using alternative models and in vitro tests for safety testing. This review will examine the current state of three alternative models - whole embryo culture (WEC), the mouse embryonic stem cell test (mEST), and zebrafish. Each of these alternatives will be reviewed, and advantages and disadvantages of each model will be discussed. These models were chosen because they are the models most commonly used and would appear to have the greatest potential for future applications in developmental toxicity screening and testing.

Applications of stem cells in developmental toxicology

Publisher Summary This chapter discusses the applications of stem cells in developmental toxicology. Three classes of stem cells have been discovered by scientists. Embryonic stem cells are pluripotent cells capable of giving rise to tissues of the developing embryo, including development of the germ line. Adult stem cells have been identified in many tissues of the adult organism where they maintain tissue homeostasis and with limitations are able to replace cells that die due to injury or disease. The stem cells are used in developmental toxicology testing which is called as embryonic stem cell test (EST). The EST was recently applied to chemicals used in the cosmetic industry; it was also utilized to determine potential developmental toxicity of four different sizes of silica nanoparticles. A number of modifications for the EST have been suggested by various authors. One possibility to reduce the number of animals used in developmental toxicity testing would be the inclusion of the EST using human cells in place of the second species required. In addition to this, culture conditions can alter the ability of stem cells to differentiate, and in fact, different additions need to be made to allow the cells to differentiate along particular pathways. Although there is great promise in the utilization of stem cells for a variety of uses, including developmental toxicity testing, there is still much work to be done and much knowledge to be gained before their role in regulatory science can be defined.

Transcript Profiling in the Testes and Prostates of Postnatal Day 30 Sprague-Dawley Rats Exposed Prenatally and Lactationally to 2-Hydroxy-4-methoxybenzophenone

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21. Gene expression profiles of the prostate and testis were differentially affected by HMB dose with significant alterations observed at the 30,000 ppm HMB group. Tissue-specific gene expression was also identified. These genes, whose expression was altered by HMB exposure, may be considered as candidate biomarker(s) for testicular or prostatic toxicity; however, further studies are necessary to explore this potential.