A Regional Meeting Continues to Grow: The 2nd Annual Michigan Alliance for Reproductive Technologies and Science Conference

Keywords::

• reproductive biology
• conference
• Michigan

The second annual Michigan Alliance for Reproductive Technologies and Science (MARTS) conference was held on the Michigan State University East Lansing campus in the spring of 2011. This forum provided an inclusive approach to the study of reproductive biology showcasing the exemplar translational and basic research efforts of reproductive scientists and clinicians throughout the State of Michigan. In this issue of SBiRM the work of several of these researchers is highlighted.

Two separate groups from the University of Michigan provided data demonstrating how traditional cell culture strategies could be augmented to better support in vitro preimplantation embryonic development. Jason Swain et al. reported on the effects of using various dipeptides as osmoregulators in culturing preimplantation mouse embryos. Though both forms of dipeptide glycine tested in this study were shown to be capable, osmolyte media which used individual amino acids to regulate osmolarity yielded higher blastocyst development rates. Notably, although ammonia levels were low across treatments, media containing dipeptide glycine produced significantly less of this byproduct than that which contained glycine alone. These researchers suggested that other as yet untested dipeptide amino acids should be assessed for their ability to serve as osmoregulators. Continuing the discussion of how to improve preimplantation culture methodologies André Monteiro da Rocha et al. from the laboratory of Gary D. Smith described how a microfluidic culture system could improve overall human embryo quality relative to a static system. Rather than as typical, growing cells in large volume liquid culture, a fixed microdrop volume device developed by this group, uses piezoelectric pins to move fluid in gentle pulses through channels connecting a media reservoir to a microfunnel which houses the embryo. This allows for the removal of waste products which build up in static systems but does not result in the complete loss of relevant biomolecules which are swept away by continual perfusion. Compared to static culture systems this device has already been shown to significantly improve embryonic development in mouse achieving implantation rates which approach that observed in vivo [Heo et al. 2010]. Applying this technology to the culturing of human embryos, this group observed a significantly reduced blastomere fragmentation rate as well as a significant increase in embryo quality compared to static cultures. Though the results obtained in mouse are encouraging it remains to be seen whether this new technology will improve blastocyst formation and pregnancy rates in humans.

A series of presenters provided the audience with both clinical and basic research perspectives on issues of female reproductive pathology. Genetic ablation of mitogen-inducible gene 6 (Mig-6) in mice is known to cause infertility and the loss of progesterone induced inhibition of estrogen stimulated uterine epithelial growth. This results in endometrial hyperplasia and tumor development [Jeong et al. 2009; Kim et al. 2010]. Expanding upon these previous studies Kim et al. from the laboratory of Jae-Wook Jeong at Michigan State University College of Human Medicine presented work which outlined the effects of overexpressing Mig-6 in a mouse endometrial cancer model lacking the phosphatase and tensin homolog (Pten) gene. Overexpression of Mig-6 completely prevented endometrial cancer development in this model system unlike the Pten knockout mice which exhibited tumor formation within 3 months. Continuing the discussion of cancers of the female reproductive tract, Fletcher-King et al. from the laboratory of Ghassan M. Saed, Wayne State University, School of Medicine, presented evidence demonstrating that shifting the redox balance in epithelial ovarian cancer (EOC) cells from anaerobic to aerobic metabolism may offer novel therapeutic strategies for the management of these types of malignancies. Chemical stimulation of pyruvate dehydrogenase altered the levels of several key oxidative stress enzymes resulting in an increase in apoptosis in EOC cells. Unlike endometrial and ovarian cancers which are comparatively prevalent in the human population Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, is relatively rare. This condition is characterized by the congenital absence of the uterus and upper section the vagina [Morcel et al. 2007]. Courtney Marsh et al. of the University of Michigan provided a retrospective chart review of 26 females with MRKH between 10 and 30 years of age. Meta-analysis of the clinical data demonstrated that pelvic pain and the presence of uterine remnants are prevalent in MRKH patients. The association between pelvic pain and the presence of uterine remnants in 70% of the MRKH cases evaluated suggested that these diagnoses may be related. Baring some similar to a clinic chart review the Contraception Health Surveillance Program, discussed by Dalen Agnew et al., Michigan State University Diagnostic Center for Population and Animal Health, has been collecting data on the reproductive tracts of zoo animals for over 20 years. This project is an invaluable resource for information relevant to contraceptive use and fertility in non-human and non-rodent species.

Another focal point of several presentations was lineage commitment in pre- and postimplantation embryos. A well know feature of embryonic stem cell (ESC) differentiation is the silencing of Nanog and Oct4. These pluripotency genes are not expressed in latter cell-types including trophoblast stem cells (TSCs). Timothy Carey et al. from the laboratory of Jason G. Knott at Michigan State University reported on their finding that the inactivation of these genes in TSCs is preceded by several key chromatin regulatory events in ESCs. These include the recruitment of several factors to the enhancers of these genes in ESCs along with the concomitant accumulation of histone H3 in place of modified histones correlated with gene activation. These changes coincided with a reduction in chromatin accessibility as well as the adoption of a DNA methylation pattern resembling that observed in TSCs. These results provide a strong foundation for further inquiries into how pluripotency genes are functionally silenced during TSC differentiation. Two separate presenters from the Rappolee laboratory at Wayne State University School of Medicine focused on the impact of stress and stress enzymes in both ESCs and TSCs.

Before implantation the embryo consists entirely of ESCs and TSCs. Within two days of implantation, a population of TSCs must differentiate into hormone-secreting trophoblast giant cells (TGCs) while a portion of the ESCs must differentiate into nutrient-acquiring endoderm cells. These researchers presented data supporting their theory that these events are regulated by stress in a dose-dependent manner. The proposed model therefore predicts that the ratio of markers of early versus late differentiation events will increase in response to pathophysiological or toxicological stressors. As TSC differentiation proceeds specific subpopulations of mature trophoblasts arise relative to their position within the blastocyst. The more peripherally located extravillous trophoblasts (EVTs) undergo a process termed integrin-switching. This exchange of integrin proteins along the cell surface is required for normal invasion of the endometrium and is stimulated by exposure to Heparin-binding EGF-like growth factor (HBEGF) [Jessmon et al. 2010]. Philip Jessmon et al. from the laboratory of David R. Armant, Wayne State University School of Medicine, expanded upon this finding by demonstrating that the mRNA levels of the proteins undergoing integrin switching in cytotrophoblast cells are not altered following HBEGF exposure demonstrating that this process is regulated post-transcriptionally. Further, when this group cultured cytotrophoblasts on Matrigel (MG) normal integrin switching proceeded in the absence of HBEGF. However, this activity was blocked following the depletion of growth factors from the MG. This inhibited trophoblast invasion but the activity of the cells could be rescued by subsequent treatment with HBEGF. Together these results provide further evidence for the necessity of integrin switching during cytotrophoblast invasion and suggest that induction of this process may exhibit a degree of redundancy.

Not all of the research presented at MARTS concerned female reproductive health and embryonic development. Mature mammalian spermatozoa have long been known to lack full length ribosomal RNAs (rRNAs). Graham Johnson et al. from the laboratory of Stephen A. Krawetz, Wayne State University School of Medicine, presented data which countered the prevailing opinion that the absence of intact 18S and 28S rRNAs is due to the expulsion of bulk cytoplasm during sperm maturation. Characterization of the RNA population retained in mature sperm by Next Generation Sequencing clearly demonstrated that fragments of the rRNAs are the most abundant transcripts found in the mature male gamete. Approximately 80% of sequencing reads aligned to these transcripts resulting in a level of representation approaching that observed in somatic cells. Subsequent analysis of the 28S RNA by RT-PCR demonstrated that specific regions of this transcript were absent in sperm though abundant in testis which possess intact rRNAs. This observation in conjunction with the abundance of rRNA fragments detected in mature sperm suggests that translational cessation in the mature sperm is assured by the selective cleavage of the 18S and 28S transcripts [Johnson et al. 2011].

Each of the three oral platforms at the MARTS conference began with a presentation by a leading reproductive biologist from one of the three major research universities in the State of Michigan. George Smith highlighted various research programs underway within the Reproductive and Developmental Science Program at Michigan State University. Representing Wayne State School of Medicine, Derek Wildman discussed the evolution of pregnancy and placentation in mammals. Lastly, Gary Smith from the Reproductive Sciences Program at the University of Michigan introduced the growing number of stem cell derivation projects and collaborations within the State of Michigan.

The MARTS conference culminated with the keynote address delivered by Renee Reijo Pera, a world renowned leader in the study of human embryo growth and development. In addition to supervising a highly productive laboratory Dr. Reijo Pera serves as the Director of the Human Embryonic Stem Cell Research and Education at Stanford University. Her presentation highlighted the results of a recent study completed by her group which was published in Nature Biotechnology [Wong et al. 2010]. This work used a novel noninvasive time-lapse imaging approach to monitor the development of human zygotes generated by in vitro fertilization. The ability of these embryos to reach the blastocyst stage and the quality of the resulting blastocyst were able to be predicted by measuring the length of the first cytokinesis, and the amount of time spent between the first and second, and the second and third mitoses. Embryos which failed to progress within a defined window of time exhibited perturbed gene expression levels compared to those which were predicted to develop into high quality blastocysts. These results are expected to be highly relevant to assisted reproduction clinics which often rely on determining the quality of an embryo well after it has progressed past the third mitosis.

This issue of SBiRM contains two invited manuscripts each of which encapsulates a single issue presented at the MARTS conference. The first of these studies examines the incidence of chromosomal numerical abnormalities in sperm produced by carriers of a chromosome structural rearrangement. Ester Anton et al. from the Universitat Autònoma de Barcelona focused on the three most frequently observed reorganization events in humans, Robertsonian translocations, reciprocal translocations, and inversions, as well as a smaller subset of carriers exhibiting complex chromosomal rearrangements [Anton et al. 2011]. Of the 136 patients examined more than 45% of these individuals were considered at risk of producing aneuploid or diploid gametes relative to the general population. Within the noncomplex groups of structural rearrangements the Robertsonian and reciprocal translocation carriers produced gametes exhibiting numerical abnormalities at a much higher frequency than that observed in the inversion group. However, sperm from the complex chromosome rearrangement carriers were the most affected. Greater than 70% of these individuals exhibited elevated levels of gametes with abnormal chromosome numbers.

The second invited manuscript examined the relationship between hormonal signaling and tumor suppressor proteins in breast cancer. Though the estrogen metabolite 2-methoxyestradiol (2-ME₂) is known to induce apoptosis in a variety of tumors how this anti-proliferative effect is achieved remains to be elucidated. Following the depletion of endogenous steroids from cultured breast cancer cells Amy Siebert et al. from the laboratory of Virinder K. Moudgil at Oakland University examined the relative expression levels of the tumor suppressor protein p53 upon exposure to a range of 2-ME₂ concentrations [Siebert et al. 2011]. The authors observed increased expression of p53 following treatment with 0.01 – 1 µM 2-ME₂. This was accompanied by a dramatic reduction in cell number and dose dependent changes in the cellular localization of p53. Future studies will address the mechanism by which 2-ME₂ contributes to the regulation of this tumor suppressor protein.

The breadth of topics discussed at the second annual MARTS meeting complemented the vast number of subfields found within the reproductive sciences. This inclusive approach towards scientific exchange will be built upon in the coming years to extend the impact of this forum beyond the State of Michigan. In addition we hope to attract individuals who straddle the divide between the laboratory and the clinic by hosting both a basic research and a clinical keynote speaker. We believe that by maintaining the high level of excellence exhibited during our past two meetings we will be able to establish this event as the premiere reproductive biology conference in the American Midwest within a few short years. However, this goal cannot be achieved without your support. The 2012 MARTS organizing committee would like to invite you to attend our third annual conference to be held on May 23, 2012. This event will be hosted by Wayne State University at the Detroit Yacht Club on Belle Isle. Please visit our website for registration information as well as other important updates (http://compbio.med.wayne.edu/MARTS2012.html). We look forward to seeing you next year.

Abbreviations

MARTS:	=	Michigan Alliance for Reproductive Technologies and Science;
Mig-6:	=	mitogen-inducible gene 6;
Pten:	=	phosphatase and tensin homolog;
EOC:	=	epithelial ovarian cancer;
MRKH:	=	Mayer-Rokitansky-Kuster-Hauser syndrome;
ESC:	=	embryonic stem cell;
TSC:	=	trophoblast stem cell;
TGC:	=	Trophoblast Giant Cell;
EVT:	=	extravillous trophoblasts;
HBEGF:	=	heparin-binding EGF-like growth factor;
MG:	=	Matrigel;
rRNA:	=	ribosomal RNA;
2-ME2:	=	2-methoxyestradiol.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.