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Crystal Gomez, Arthur Gutierrez.

University of Colorado, Anschutz Medical Campus, Aurora, CO.

Pituitary somatotropes and lactotropes, expressing GH and PRL respectively, retain plasticity, allowing rapid cell expansions in response to increased physiological demands. For example, the somato-lactotrope cell expands into lactotropes during pregnancy and into somatotropes with exercise. Unfortunately, very little is known about the signaling events that instruct somato-lactotrope cells to proliferate and then terminally differentiate or that contribute to the transformed phenotype. In vivo transgenic mouse studies revealed that uncontrolled activation of growth factor Ras/MAPK signaling pathways in lactotropes results in lactotrope hyperplasia. Moreover, in vitro studies using GH4 and GH3 rat somato-lactotrope tumor cell lines have shown that pMAPK is necessary for both short-term proliferation and differentiation, with the duration of pMAPK activation possibly dictating differential responses. However, these proliferation studies all relied on short-term (6 - 24 h) assays. Thus, the specific role of MAPK in durable lactotrope proliferation and differentiation responses (i.e., cell counts and phenotypic characterization over about 5 days), remain unknown. Furthermore, the role of mTOR signaling in either of these as durable responses is not well understood. In order to directly interrogate the role of EGF and mTOR, we used persistent EGF addition or pharmacological inhibitors to interrogate the role of these pathways in GH4 cell proliferation and clonogenicity. Using long-term proliferation and clonogenicity assays, we found that treating GH4 cells with EGF reduced cell proliferation and clonogenicity.

Collectively, these data reveal that the EGF-signaling pathway, via Ras/Raf/MAPK, serves to promote lactotrope differentiation, whereas activation of the PI3K/mTOR/S6K pathway regulates lactotrope proliferation and transformed phenotype.


Biological Sciences FRI-515



Alejandra Santiago, Catharina Lindley-Casper, William Sullivan.

University of California, Santa Cruz, Santa Cruz, CA.

Wolbachia is a bacterial endosymbiont that is efficiently transmitted through the female germ line in millions of insect species. Wolbachia is the cause of river blindness and elephantiasis, diseases afflicting over 200 million people worldwide. Similar to other pathogens, Wolbachia is surrounded by a host membrane. Rab GTPases play a role in cellular trafficking of these pathogens. Because of this, we hypothesized that Rabs, which are proteins involved in the regulation of membrane transport, may also participate in Wolbachia biology either by direct transport of the bacteria or by transport of nutrients for bacterial growth. Previous studies demonstrate that Wolbachia rely on microtubules and motor proteins for efficient transport into the oocyte from the neighboring nurse cells in Drosophila melanogaster female ovaries. Using microscopy, we demonstrate that Wolbachia transport also relies indirectly on membrane trafficking pathways. We find that Wolbachia transport from the nurse cells into the oocyte is reduced in ovaries expressing transgenic YFP-Rab6, a small GTPase specifically required for Golgi-to-ER membrane trafficking. We are investigating whether this ectopic expression is similar to expression in oocytes of infected mutant Rab6 lines.

The results will allow us to better understand the role Rab6 plays in the distribution of Wolbachia throughout oocyte development. This finding has important implications for developing new drugs to target the bacteria.



IN MICE Rose Joachim, Ryan Haley, Brian Hermann.

University of Texas at San Antonio, San Antonio, TX.

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man’s life. Transplanted SSCs have been shown to regenerate spermatogenesis in a variety of animal models, and most recently, in rhesus macaques that were made infertile due to high-dose busulfan chemotherapy. In that recent study, the hematopoietic deficits induced by high-dose busulfan chemotherapy were addressed using autologous transplants of granulocyte colonystimulating-factor (G-CSF)-mobilized hematopoietic stem cells. While this regimen reconstituted each animal’s hematopoietic system, a meta-analysis revealed that G-CSF treatment surprisingly protected rhesus spermatogenesis from busulfan-induced infertility. Thus, G-CSF may be able to prevent infertility after chemotherapy and obviate the need for risky procedures like SSC transplantation. Similar G-CSF treatments in mice led to significantly better recovery of spermatogenesis after busulfan treatment than in untreated controls, suggesting enhanced SSC survival.

To determine if G-CSF treatment caused increased survival of undifferentiated spermatogonia (including SSCs) after busulfan treatment, we compared the testes of mice treated with busulfan with and without G-CSF 4 days after busulfan treatment. Testis sections were costained for PLZF (a marker of undifferentiated spermatogonia) and either activated caspase3 or TUNEL (apoptotic markers) are currently being analyzed. Our initial results demonstrate that testes from busulfan/G-CSF-treated mice contain more PLZF+ cells than testes of mice treated with busulfan alone. Although our analysis is ongoing, these preliminary results suggest G-CSF promotes survival of PLZF+ spermatogonia after busulfan treatment. Ultimately, this work may solidify a new fertility-sparing treatment that will circumvent the need for risky interventions like SSC transplantation.



Steven Anzaldua, Brandi Betts-Obregon, Andrew Mendiola, Jeff Grigsby, Andrew Tsin.

University of Texas at San Antonio, San Antonio, TX.

Diabetic retinopathy (DR) is characterized by the leakage of blood into the eye in its nonproliferative stage and by hypoxia-induced angiogenesis in the retina in the more advanced proliferative stage. This in turn will cause blood to leak into the vitreous humor resulting in more complications such as retinal detachment and blindness. It is well documented that young women and pregnant women have fluctuating levels of estrogen in the body which is known to exacerbate DR. Although no cure for DR currently exists, the purpose of this study is to further our understanding of the mechanisms involved in the progression of DR. The effects of various estrogen concentrations on rhesus monkey retinal endothelial cells (RhREC) will be tested. Moreover, we hypothesize that the viability of a continuous cell line of RhREC will increase under hyperglycemic estrogen conditions but not under hyperglycemic conditions alone. RhREC


will be tested in vitro under hyperglycemic conditions using a glucose concentration of 18.5 mM (versus a control concentration of 5.5 mM) along with estrogen (E2) at 0.0, 0.1, 1.0, and 10.0 nM. Cell viability will be tested over 72 hours with interval time points every 24 hours along with imaging of cell morphology to determine if E2 + 18.5 mM glucose effects are dose and time dependent. This is a novel approach, attempting to show a relationship between high glucose and exogenous estrogen effects on RhREC. Results from our experiments will provide important information on the development of progressive DR.



Eden Barragan, Jacqueline Algara, Soleil Schutte, Ryan Schutte, Diane O’Dowd.

University of California, Irvine, Irvine, CA.

Mutations in the voltage gated sodium channel gene SCN1A cause a wide spectrum of epilepsy disorders, from the mild form of genetic epilepsy with febrile seizures plus (GEFS+) to the severe form of Dravet syndrome (DS). GEFS+ is characterized by childhood-onset febrile seizures that persist beyond 6 years. In DS, seizures appear within the first year of life, can lead to cognitive impairment by the age of 2, and are often resistant to pharmacotherapy. We used 2 knock-in fly lines, one with a GEFS+ (K1270T) mutation and 1 with a DS (S1231R) mutation, as model systems to search for new therapies. Consistent with disease symptoms in humans, the GEFS+ and DS flies exhibit heat-induced seizures and the seizure phenotype is more severe in DS compared to GEFS+ flies. Our initial studies focused on the monoamine signaling since we found that seizure sensitivity was altered in DS and GEFS+ flies in a genetic background (white eyes) that affects monoamine levels. Feeding flies the serotonin precursor, 5-hydroxytryptophan (5-HTP) significantly reduces seizure sensitivity in DS mutants, but increases seizures in GEFS+ mutants. In contrast, a dopamine synthesis inhibitor, 3-iodotyrosine (3-IY), significantly reduces seizure sensitivity in GEFS+ mutants, but marginally reduces seizure sensitivity in DS flies. Neither GEFS+ nor DS flies showed a significant difference in seizure activity when fed histamine or dopamine. These data suggest that the serotonin and dopamine pathways regulate DS and GEFS+ seizure phenotypes, suggesting alternative therapeutic targets for the treatment of epileptic disorders.




Jennifer Ausland, Magesh Thiyagarajan, Xavier Gonzales.

Texas A&M University-Corpus Christi, Corpus Christi, TX.

Bacillus cereus, Salmonella enterica, and Vibrio vulnificus are among common species causing foodborne outbreaks in the United States. This study investigates effectiveness of cold plasma in sterilizations of B. cereus vegetative cells and spores; S. Typhimurium on eggs shells, chicken meat, tomato, and papaya; and V. vulfinicus on live oysters. Plasma was applied to B. cereus vegetative cells on tryptic soy agar plates (TSA) for various exposure times, incubated, and inactivation recorded. B. cereus spores were dispersed on Petri plates, treated for 360 s, and suspended to determine inactivation. Plasma was applied to Salmonella on sample surfaces for various times, suspended, and plated to record inactivation. Plasma was then applied to V. vulnificus on agar oysters. Surfaces of treated oysters were swabbed and plated. Meat of treated oysters was homogenized and plated. Oyster surface and internal inactivation was recorded. Results confirmed 99% inactivation of B. cereus vegetative cells after 90 s and

0.84 log reduction of spores after 360 s. Inactivation of Salmonella on agar was 1.6 logs after 120 s, 0.49 logs on chicken eggs after 360 s, 1.1 logs on chicken meat after 360 s, 2.6 logs on tomato after 360 s, and 1.7 logs on papaya after 360 s. Lastly, V. vulnificus was completely eliminated on agar after 180 s, completely eliminated on oyster surface after 120 s, and reduced by one fold on internal homogenate of oyster after 360 s. Results show that cold plasma can be an alternative sterilization method in the food industry.



Nancy Pina, Brian Hermann.

University of Texas at San Antonio, San Antonio, TX.

In mammals, spermatogenesis occurs in the seminiferous tubules in the testes, and is dependent on the continual self-renewal and differentiation of spermatogonial stem cells (SSCs). The characteristics and extent of the SSC pool in primate species remains the subject of intense scientific debate. Undifferentiated spermatogonia in primate


Biological Sciences testes include Adark and Apale spermatogonia, which have been designated reserve and renewing SSCs, respectively.

However, a male germline stem cell system with two distinct stem cells would be unique among mammals, and recent data suggests Adark and Apale share many phenotypic characteristics. A recent study in mice revealed inhibitor of DNA binding 4 (ID4) is exclusively expressed by Asingle SSCs and is restricted to stem cells in the testes. Thus, assuming that the characteristics of rodent spermatogenesis are conserved in primates, ID4 may be a useful marker to define the extent of the SSC pool in primates and resolve the debate about whether Adark and/or Apale spermatogonia are the bona fide SSC in primate testes. Thus, we are examining ID4 expression in rhesus, macaque, and baboon testes using immunohistochemistry to determine whether Adark and/or Apale spermatogonia label for this marker. While this work is currently ongoing, we expect the results will give us insight into the identity of the primate SSC, which has important implications for understanding the basic biology of primate spermatogenesis and leading to clinical techniques such as SSC transplantation to regenerate spermatogenesis in infertile testes.



Christie Rodriguez-Ramirez, Kimberly Kremer, Karen Hedin.

Mayo Clinic Graduate School of Medicine, Rochester, MN.

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