FLARR Pages #44: Uses of iMovie

Need a new idea to involve technology and fun? Using the Macintosh iMovie program or other movie making software, students can create movies in Spanish. You can use any topic to make a movie and the choice aspect is very attractive to students

Perturbations in Lineage Specification of Granulosa and Theca Cells May Alter Corpus Luteum Formation and Function

Anovulation is a major cause of infertility, and it is the major leading reproductive disorder in mammalian females. Without ovulation, an oocyte is not released from the ovarian follicle to be fertilized and a corpus luteum is not formed. The corpus luteum formed from the luteinized somatic follicular cells following ovulation, vasculature cells, and immune cells is critical for progesterone production and maintenance of pregnancy. Follicular theca cells differentiate into small luteal cells (SLCs) that produce progesterone in response to luteinizing hormone (LH), and granulosa cells luteinize to become large luteal cells (LLCs) that have a high rate of basal production of progesterone. The formation and function of the corpus luteum rely on the appropriate proliferation and differentiation of both granulosa and theca cells. If any aspect of granulosa or theca cell luteinization is perturbed, then the resulting luteal cell populations (SLC, LLC, vascular, and immune cells) may be reduced and compromise progesterone production. Thus, many factors that affect the differentiation/lineage of the somatic cells and their gene expression profiles can alter the ability of a corpus luteum to produce the progesterone critical for pregnancy. Our laboratory has identified genes that are enriched in somatic follicular cells and luteal cells through gene expression microarray. This work was the first to compare the gene expression profiles of the four somatic cell types involved in the follicle-to-luteal transition and to support previous immunofluorescence data indicating theca cells differentiate into SLCs while granulosa cells become LLCs. Using these data and incorporating knowledge about the ways in which luteinization can go awry, we can extrapolate the impact that alterations in the theca and granulosa cell gene expression profiles and lineages could have on the formation and function of the corpus luteum. While interactions with other cell types such as vascular and immune cells are critical for appropriate corpus luteum function, we are restricting this review to focus on granulosa, theca, and luteal cells and how perturbations such as androgen excess and inflammation may affect their function and fertility

Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress

84% of the population will suffer from low back pain at some point in their lives, and 39% of the cases may be related to pain originating from the intervertebral disc (IVD) also known as discogenic pain. Age or injury can cause progressive degeneration of the IVD, and this can allow innervation by nociceptive (pain-sensing) neurons from the dorsal root ganglion (DRG) into the inner IVD.6 Long-term exposure to pain-inducing stimuli can cause nociceptor sensitization, manifested as increased pain sensation in response to non-painful or painful stimuli.7 Sensitization results in the nociceptors responding more strongly to a stimulus, and the activation energy required for response is reduced.8 During IVD degeneration, inflammation and oxidative stress create excess reactive oxygen species (ROS) .9 ROS such as superoxide may play a role in nociceptive signaling and lead to nociceptor sensitization.8 ROS are involved in the chronic pain that results from nerve injury or inflammation in many contexts and may be involved in discogenic pain as well.10 The focus of this study is to determine if long-term oxidative stress causes a change in the DRG nociceptive neuron phenotype related to sensitization. This would increase the expression of pain-related ion channels and lower the neuronal activation threshold. 1. DePalma et al. (2011) 2. Balague et al. (2012) 3. Zhang et al. (2010) 4. Humzah et al. (1988) 5. Ohtori et al. (2015) 6. Yang et al. (2018) 7. Woolf et al. (2010) 8. Chung et al. (2004) 9. Nasto et al. (2013) 10. Yowtak et al. (2011

A Tunable, Three-Dimensional \u3ci\u3eIn Vitro\u3c/i\u3e Culture Model of Growth Plate Cartilage Using Alginate Hydrogel Scaffolds

Defining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long- and short-range signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroid-related protein–Indian hedgehog (PTHrP-IHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell proliferation and inhibited hypertrophy, whereas IHH signaling stimulated chondrocyte hypertrophy. Importantly, the treatment of alginate bead cultures with IHH or thyroxine resulted in formation of a discrete domain of hypertrophic cells that mimics tissue architecture of native growth plate cartilage. Together, these studies are the first demonstration of a tunable in vitro system to model the signaling network interactions that are required to induce zonal architecture in growth plate chondrocytes, which could also potentially be used to grow cartilage cultures of specific geometries to meet personalized patient needs

Letrozole: A Steroid-Free Estrous Synchronization Method

Most bovine estrous synchronization protocols utilize progesterone plus estrogen to control ovulation timing. A drug that inhibits estrogen production (Letrozole) may be an alternative, steroid-free synchronization method (not yet commercially available). However, low estrogen can negatively affect the health of follicles/oocytes and impact fertility. To determine its effects, Letrozole was administered intramuscularly while tracking follicle growth and circulating hormones. Letrozole response was variable. Two of three cows experienced delayed luteolysis/ovulation and extended progesterone production. This preliminary data indicates that Letrozole treatment allows normal follicle progression but drug response may vary and little is known about effects on oocyte quality

Transcriptomic data of bovine ovarian granulosa cells of control and High A4 cows

Microarray analysis using Affymetrix Bovine GeneChip 1.0 ST Array to determine RNA expression analysis was performed on somatic granulosa cells from two different groups of cows classified based on androstenedione concentration within the follicular fluid (Control vs High A4) of estrogen-active dominant follicles. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE97017 - RNA Expression Data from Bovine Ovarian Granulosa Cells from High or Low Androgen-Content Follicles). Subsequent ANOVA determined genes that were enriched (≥ 1.5 fold more) or decreased (≤ 1.5 fold less) in the High A4 granulosa cells compared to Control granulosa cells and analyzed filtered datasets of these differentially expressed genes are presented as tables. MicroRNAs that are differentially expressed in Control and High A4 granulosa cells are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values are shown as a figure. Ingenuity Pathway Analysis determined upstream regulators of differently expressed genes as presented in a table. These data have been further analyzed and interpreted in the companion article “A High-Androgen Microenvironment Inhibits Granulosa Cell Proliferation and Alters Cell Identity.

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these four cell types. Analysis of the RNA present in each bovine cell type using Affymetrix microarrays yielded new cell-specific genetic markers, functional insight into the behavior of each cell type via Gene Ontology Annotations and Ingenuity Pathway Analysis, and evidence of small and large luteal cell lineages using Principle Component Analysis. Enriched expression of select genes for each cell type was validated by qPCR. This expression analysis offers insight into cell-specific behaviors and the differentiation process that transforms somatic follicular cells into luteal cells

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide

Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress

84% of the population will suffer from low back pain at some point in their lives, and 39% of the cases may be related to pain originating from the intervertebral disc (IVD) also known as discogenic pain. Age or injury can cause progressive degeneration of the IVD, and this can allow innervation by nociceptive (pain-sensing) neurons from the dorsal root ganglion (DRG) into the inner IVD.6 Long-term exposure to pain-inducing stimuli can cause nociceptor sensitization, manifested as increased pain sensation in response to non-painful or painful stimuli.7 Sensitization results in the nociceptors responding more strongly to a stimulus, and the activation energy required for response is reduced.8 During IVD degeneration, inflammation and oxidative stress create excess reactive oxygen species (ROS) .9 ROS such as superoxide may play a role in nociceptive signaling and lead to nociceptor sensitization.8 ROS are involved in the chronic pain that results from nerve injury or inflammation in many contexts and may be involved in discogenic pain as well.10 The focus of this study is to determine if long-term oxidative stress causes a change in the DRG nociceptive neuron phenotype related to sensitization. This would increase the expression of pain-related ion channels and lower the neuronal activation threshold. 1. DePalma et al. (2011) 2. Balague et al. (2012) 3. Zhang et al. (2010) 4. Humzah et al. (1988) 5. Ohtori et al. (2015) 6. Yang et al. (2018) 7. Woolf et al. (2010) 8. Chung et al. (2004) 9. Nasto et al. (2013) 10. Yowtak et al. (2011