Alla Bondareva

The Lysyl Oxidase Inhibitor, β-Aminopropionitrile, Diminishes the Metastatic Colonization Potential of Circulating Breast Cancer Cells

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (μCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention.

1H NMR metabolomics identification of markers of hypoxia-induced metabolic shifts in a breast cancer model system

Hypoxia can promote invasive behavior in cancer cells and alters the response to therapeutic intervention as a result of changes in the expression many genes, including genes involved in intermediary metabolism. Although metabolomics technologies are capable of simultaneously measuring a wide range of metabolites in an untargeted manner, these methods have been relatively under utilized in the study of cancer cell responses to hypoxia. Thus, 1H NMR metabolomics was used to examine the effects of hypoxia in the MDA-MB-231 human breast cancer cell line, both in vitro and in vivo. Cell cultures were compared with respect to their metabolic responses during growth under either hypoxic (1% O2) or normoxic conditions. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify a set of metabolites that were responsive to hypoxia. Via intracardiac administration, MDA-MB-231 cells were also used to generate widespread metastatic disease in immuno-compromised mice. Serum metabolite analysis was conducted to compare animals with and without a large tumor burden. Intriguingly, using a cross-plot of the OPLS loadings, both the in vitro and in vivo samples yielded a subset of metabolites that were significantly altered by hypoxia. These included primarily energy metabolites and amino acids, indicative of known alterations in energy metabolism, and possibly protein synthesis or catabolism. The results suggest that the metabolite pattern identified might prove useful as a marker for intra-tumoral hypoxia.

Viral transduction of male germline stem cells results in transgene transmission after germ cell transplantation in pigs.

ABSTRACT Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo‐derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES‐like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA‐1, and SSEA‐4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5–6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells. Mol. Reprod. Dev. 78:202–211, 2011.

Phthalate esters affect maturation and function of primate testis tissue ectopically grafted in mice

Di-n-Butyl (DBP) and Di-(2-EthylHexyl) (DEHP) phthalates can leach from daily-use products resulting in environmental exposure. In male rodents, phthalate exposure results in reproductive effects. To evaluate effects on the immature primate testis, testis fragments from 6-month-old rhesus macaques were grafted subcutaneously to immune-deficient mice, which were exposed to 0, 10, or 500 mg/kg of DBP or DEHP for 14 weeks or 28 weeks (DBP only). DBP exposure reduced the expression of key steroidogenic genes, indicating that Leydig cell function was compromised. Exposure to 500 mg/kg impaired tubule formation and germ cell differentiation and reduced numbers of spermatogonia. Exposure to 10 mg/kg did not affect development, but reduced Sertoli cell number and resulted in increased expression of inhibin B. Exposure to DEHP for 14 week also affected steroidogenic genes expression. Therefore, long-term exposure to phthalate esters affected development and function of the primate testis in a time and dosage dependent manner.

Lymphoid-Specific Helicase (HELLS) Is Essential for Meiotic Progression in Mouse Spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells−/− to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells−/−, Hells+/−, and Hells+/+ mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells−/− than from Hells+/+ mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells−/− mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells−/− spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.

Expression pattern of acetylated α-tubulin in porcine spermatogonia

Mammalian spermatogonial stem cells reside on the basement membrane of the seminiferous tubules. The mechanisms responsible for maintenance of spermatogonia at the basement membrane are unclear. Since acetylated α‐tubulin (Ac‐α‐Tu) is a component of long‐lived, stable microtubules and deacetylation of α‐tubulin enhances cell motility, we hypothesized that acetylation of α‐tubulin might be associated with positioning of spermatogonia at the basement membrane. The expression pattern of Ac‐α‐Tu at different stages of testis development was characterized by immunohistochemistry for Ac‐α‐Tu and spermatogonia‐specific proteins (PGP 9.5, DAZL). In immature pig testes, Ac‐α‐Tu was present exclusively in gonocytes at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating toward the tubule periphery and Ac‐α‐Tu appeared polarized toward the basement membrane. In adult pig testes, Ac‐α‐Tu was detected in few single or paired spermatogonia at the basement membrane as well as in spermatids and spermatozoa. Only undifferentiated (DAZL−), proliferating (determined by BrdU incorporation) spermatogonia expressed high levels of Ac‐α‐Tu. Comparison with the expression pattern of β‐tubulin and tyrosinated α‐tubulin confirmed that only Ac‐α‐Tu is specific to germ cells. The unique pattern of Ac‐α‐Tu in undifferentiated germ cells during postnatal development suggests that posttranslational modifications of microtubules may play an important role in recruiting and anchoring spermatogonia at the basement membrane. Mol. Reprod. Dev. 77: 348–352, 2010.

Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation†

Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor‐derived transgenic sperm. The objectives of this study were to explore a non‐viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8–9 weeks post‐irradiation. Donor cells were collected from testes of 9‐week‐old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta‐casein promoter (GBC) and a chicken beta‐globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non‐transfected cells to a total of 1.5 × 108 cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound‐guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof‐of‐concept that non‐viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255–261, 2012.

TALEN-mediated gene targeting in porcine spermatogonia

Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH‐L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non‐homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.

Goat Embryonic Stem-Like Cell Derivation and Characterization

Embryonic stem (ES) cells are derived from the inner cell masses of preimplantation embryos. ES cells are pluripotent cells with the capacity for long-term propagation and broad differentiation plasticity. These cells have an exceptional functional feature in that they can differentiate into all tissues and organs, including germ cells. Established ES cell lines have been generated in mouse, human, and nonhuman primate but derivation of ES cells in farm animals has been problematic. Several ES-like cell lines from farm animals have been reported to exhibit properties of pluripotency in vitro. However, only a few of them morphologically resemble ES cells, or express markers that are associated with established ES cell lines from mouse and humans. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. In this chapter, we describe methods for isolation of goat ES (gES) cell lines from in vivo-derived blastocysts and characterization of markers indicative of pluripotency. In addition, we outline differentiation of gES cells into all three germ layers in vivo by forming teratomas as a hallmark of pluripotency.