Obesity is a leading health issue with a negative effect on fertility, but the complexity of the relationship between obesity and fertility requires further study. Santoro and colleagues have characterized the metabolic environment of obesity and the development of reprometabolic syndrome. Due to the reproductive similarities between ewes and women, we use ovariectomized ewes as a translational model of reprometabolic syndrome and tested whether obesity alters the estradiol-17β (E2)-induced luteinizing hormone (LH) surge. Each ovariectomized ewe serves as her own biological control during the three phases of the study: normal weight (phase 1), obese (phase 2), and a return to normal weight (phase 3). Ewes were weighed weekly and 12th rib fat thickness was measured as a proxy for body condition score. During each of the three phases of the study, following an intramuscular injection of 25 μg of E2, serial blood samples were collected, and serum LH concentrations were measured by radioimmunoassay. The peak concentration and timing of the LH surge are significantly lower and delayed in obese ewes in phase 2 as compared to phases 1 and 3. Our results suggest that the mechanisms that underlie the pathological effects of obesity and reprometabolic syndrome on the E2-induced LH surge are similar in ewes and women. Therefore, ewes can serve as a model for parsing the relative roles of diminished hypothalamic and pituitary sensitivity to E2, resulting in a reduced and delayed LH surge associated with obesity and reprometabolic syndrome in women.

The Veterinary Perspectives Institute (VPI) was designed to address the challenges faced by underrepresented youth who are interested in veterinary medicine. This study examines the VPI, a tuition-free program that introduces students from under-resourced high schools to veterinary medicine and One Health through a hands-on, transdisciplinary approach. The VPI integrates One Health concepts by linking human, animal, and environmental health in the context of veterinary medicine. The program’s aim is to foster college readiness and career exploration in veterinary and biomedical fields, while also addressing barriers to diversity in veterinary medicine. Through hands-on learning and case studies in One Health, students gain exposure to STEM topics like anatomy, microbiology, and epidemiology. The program addresses systemic barriers such as limited animal exposure and lack of mentorship by pairing students with university mentors from diverse backgrounds who guide them in building academic portfolios and developing professional skills. The program also emphasizes financial literacy, mental health, and work-life balance, providing students with the tools to navigate future challenges. Daily “community circles” foster socio-emotional growth, resilience, and belonging, helping students confront impostor syndrome. Program outcomes, assessed through pre- and post-surveys, show increased awareness of educational pathways and One Health concepts. While some participants reconsidered careers in veterinary medicine, the program successfully cultivated a college-going mindset and interest in STEM careers. By combining mentorship, transdisciplinary learning, and community building, the VPI offers a replicable model to address diversity gaps in veterinary medicine and inspire a new generation of professionals grounded in the One Health framework.

Activin, GnRH, and estrogen are key endocrine inputs known to regulate the GnRH receptor (GnRHR) promoter; however, it has become increasingly evident that the mechanisms regulating the GnRHR promoter vary by model and species. To explore these differences, transgenic mice harboring either a wild-type mouse GnRHR (mGnRHR) or sheep (oGnRHR) GnRHR promoter fused to luciferase (-LUC) were infected with an adenovirus overexpressing follistatin, neutralizing activin and decreasing serum concentrations of FSH in both animal models. However, follistatin overexpression in the oGnRHR-LUC mouse more than doubled luciferase expression, whereas in the mGnRHR-LUC animals it led to a 40% decrease in luciferase expression. Thus, the divergent transcriptional responses of the mouse and sheep GnRHR genes to activin appear to be reliably recapitulated in transgenic mice. To further elucidate mechanisms regulating oGnRHR expression, a mouse with a mutated cyclic AMP response element (µCRE) in the proximal oGnRHR-LUC promoter was developed. Using an electrophoretic mobility shift assay, a specific and high affinity interaction of the ovine CRE with nuclear components exists, but these are not modified in the presence of E2, indicating that CRE binding protein (CREB) is necessary but not sufficient to mediate E2 input to oGnRHR expression.

Reduced hours of instruction are reported within the gross anatomy education literature. Anatomy instruction continues to be challenged with motivating and inspiring learners to value the contribution of gross anatomy knowledge to their career development alongside increased organizational demands for efficiency and effectiveness. To address these demands, this retrospective study sought to understand how the relative timing and amount of gross anatomy instruction were related to examination performance. Undergraduate and graduate students between 2018 and 2022 were assigned to three cohorts determined by enrollment in prosection-based anatomy only (n = 334), concurrent enrollment in prosection- and dissection-based anatomy in the same semester (n = 67), or consecutive enrollment in the courses one year apart (n = 43). Concurrent students had higher prosection-based anatomy examination scores than prosection-only and consecutive students. Consecutively, enrolled students outperformed concurrently enrolled students on the first two dissection examinations but showed no performance differences on the third and fourth dissection examinations. While the results on the timing and presentation of anatomical instruction were inconclusive, the results do support increased instructional time using both prosection and dissection modalities concurrently to improve performance on identification-based gross anatomy examinations.

Research has shown an inconsistent relationship between spatial abilities and learning outcomes from virtual anatomical tools. Instructors must understand this relationship to select appropriate resources for diverse learners. To identify appropriate tests for measuring spatial ability and evaluate the effectiveness of virtual anatomical resources, this study compared 96 students’ visuospatial ability (measured using the Mental Rotation Task [MRT] and Landmark Position on a Map [LPM] tests) with learning outcomes from experimental anatomy sessions and undergraduate anatomical course examinations. During experimental sessions, students took a test after a brief instructional session using one virtual resource: a monoscopic resource (e.g., digital photographs or a rotatable three-dimensional [r3D] specimen) or a stereoscopic virtual reality (VR) specimen. A negative linear relationship was found between MRT scores and students in Session B using VR with controllers ( r = –.56 to –.29), and LPM scores and students using VR ( r = –.71 to .39) and r3D ( r = –.41 to .43). There was a positive linear relationship between MRT scores and all other resources ( r = .01 to .91), and course examination scores ( r = .25 to .42, p = .05). Although the results were inconsistent, correlations were found between spatial ability and outcomes using both the MRT and LPM. The LPM might be better suited for determining which learners would benefit from VR. The results suggest that monoscopic resources best support high spatial abilities, and stereoscopic resources best support low spatial abilities. These findings support accounting for diverse learner visuospatial abilities when selecting resources.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

The equine chorionic girdle (CG) is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (Day 0). The CG cells differentiate into binucleate endometrial cup cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG), which has LH-like activity in the horse but variable LH- and FSH-like activity in other species. eCG has been utilized for these properties both in vivo for estrous cycle manipulation and in vitro. To collect this hormone, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare. Attempts to produce eCG in vitro using long-term culture of CG explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and remain genetically and phenotypically stable throughout long-term (i.e. months) culture. Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin long-term. The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. CG was collected from Day 33 equine conceptuses (n=3). CG was isolated manually using a dissection microscope and then enzymaticallydigested and cultured as organoids using human trophoblast organoid medium. A monolayer culture was performed as a control 2D culture model for comparison. CG organoids were assessed morphologically using brightfield microscopy, immunohistochemistry (IHC) with an anti-horse trophoblast antibody, and transmission electron microscopy (TEM), and all cultures were assessed physiologically by quantifying equine LH (as a proxy for eCG) via radioimmunoassay of conditioned media. The dense, trophoblast organoid phenotype, as described for human trophoblast organoids, was maintained for 5 passages, a period of 6 weeks. TEM of organoids revealed characteristic binucleate cells in both passages 1 and 4 and exhibited the presence of girdle cell processes, which have been described previously in endometrial cups in vivo. Furthermore, eCG was secreted at high concentrations (up to 67,000 ng/mL) up to passage 4 in organoids but only for passage 0 in monolayer cultures (up to 1,100 ng/mL). Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for early equine pregnancy. Funding was provided by Foundation for the Horse.

A healthy uterine environment is essential for normal mare fertility and is critical for ensuring a positive financial return on breeding in the equine industry. Approximately 20% of Thoroughbred mares undergo persistent breeding-induced endometritis (PBIE), creating a uterine environment that is unfavorable for pregnancy. Despite extensive in vivo studies, the exact mechanisms causing endometritis and the most efficacious strategies for preventing or resolving PBIE are unknown. Organoids are physiologically-relevant 3D cell cultures that allow disease processes to be studied at the cellular level and eliminate confounding factors present in vivo. A potential therapeutic for PBIE is extracellular vesicles (EVs) secreted by embryo-derived mesenchymal stem cells (EDMSCs). EVs are lipid-bound nanoparticles containing proteins, mRNAs, and miRNAs that are naturally secreted by cells for intercellular communication. We hypothesized that EVs secreted by EDMSCs have anti-inflammatory properties to prevent PBIE. Our objectives were i) to assess EVs produced by EDMSCs for the presence of anti-inflammatory miRNAs via PCR and ii) to assess transfer of the anti-inflammatory miRNAs from EVs to endometrial organoids stimulated for inflammationwith frozen-killed sperm (FKS) or lipopolysaccharide (LPS). Results demonstrated the presence of reported anti-inflammatory miRNAs miR-10a, miR-21, miR-24, miR-145, and miR-146a in EVs secreted by three different EDMSC cell lines. Then, endometrial organoids (n=3 mares) were co-cultured with EDMSC EVs or PBS (control) for 24 h and subsequently exposed to two inflammatory stimuli (FKS or LPS). After 12h of exposure to inflammatory stimuli, organoids were washed and assessed for the presence of anti-inflammatory miRNAs by PCR (data was normalized using U6 snRNA). MiR-10a and miR-24 trended to be increased in organoids treated with EDMSC EVs compared to PBS controls in groups stimulated with either FKS or LPS. Furthermore, uptake of EVs by the organoids was corroborated by the co-culture of organoids with fluorescently-labelled (PKH26) EVs. EV uptake was confirmed by presence of a fluorescence signal in recipient organoid cells. While studies are ongoing, data suggests that EDMSC EVs contain anti-inflammatory miRNAs that may be delivered to endometrial cells to prevent PBIE in mares. Funding was provided by the Grayson-Jockey Club Research Foundation.

Anatomy faculty with cadaver-based laboratory courses were presented with a significant challenge in March 2020 to create equivalent learning experiences without cadaveric access. The undergraduate domestic animal anatomy course at the Colorado State University was halfway into a 16-week semester when COVID-19 lockdown orders and the transition to remote instruction began. The new course curriculum was critically evaluated using student surveys and course outcome data. Most students (92.5%) agreed that the transition to online learning was a success; however, students who valued face-to-face lectures prior to March were less likely to perceive the transition as a success. Qualitative and quantitative analyses of survey results suggest that the resources perceived as most helpful for the transition to online learning were not the same as those that helped facilitate animal anatomy learning. Most students (92.5%) agreed that the Virtual Animal Anatomy (VAA) helped them learn anatomy, and 82.2% indicated that the VAA was a valuable resource following the transition to online learning. Additional resources associated with transition success included course instructors, weekly quizzes, written descriptions of anatomical structures and open laboratory sessions. In contrast, those resources associated with facilitating learning included guided quizzes and asynchronous lecture recordings. These findings suggest that the VAA can support online anatomy learning when used in conjunction with other best practices for online teaching.

Innovative reforms in medical education will require instructional tools to support these changes and to give students more flexibility in where and how they learn. At Colorado State University, the software program Virtual Canine Anatomy (VCA) was developed to assist student learning both inside and outside the anatomical laboratory. The program includes interactive anatomical photographs of dissected canine cadavers, dissection instructions with accompanying videos and diagrams, radiographs, and three-dimensional models. There is a need to evaluate the effectiveness of instructional tools like VCA so that decisions on pedagogical delivery can be evidence-based. To measure the impact of VCA on student outcomes in a dissection laboratory, this study compared student attitudes, quiz scores, dissection quality and accuracy, and instructor reliance between students with and without access to VCA. Students with VCA needed less time with teaching assistants (P < 0.01), asked teaching assistants fewer questions (P = 0.04), felt that the dissection was easier (P = 0.02), and were in stronger agreement that they had access to adequate resources (P = 0.02). No differences were found in the dissection quality or accuracy, quiz scores, or attitudes regarding overall enjoyment of the activity between the two groups. This study shows that VCA increases student independence and can be used to enhance anatomical instruction.

Orthotic devices are an established treatment for neuromusculoskeletal disease in the human population. Orthoses are an emerging veterinary therapy due to limited practitioner experience, availability of devices, and published data from veterinary patient outcomes. Expanding client education and veterinary expertise in the application of orthoses may allow greater access and successful utilization of these devices to treat appendicular disease. While orthoses have the potential to improve quality of life for veterinary patients, consideration needs to be made for owner related factors with device use. Owner satisfaction and experience may greatly impact compliance with treatment recommendations; therefore, it is crucial that owner expectations are met. The purpose of the present study was to evaluate owner-reported outcomes of orthosis for canine patients and their owner's subjective responses about the shared pet/owner experience utilizing a promoter score. It was hypothesized that owner's impressions of their pet's experience with the orthotic device would influence owner perceptions of quality of life for both the owner and the pet, and these factors would impact the likelihood of the owner to recommend a veterinary orthosis to a friend. An anonymous online survey was sent to 136 clients of a single veterinary orthoses manufacturer. Fifty-six surveys were completed and included for analysis. The owner's reported quality of life was in agreement (P = 0.02) with reported pet quality of life. There was also a higher likelihood (P = 0.02) for the owner to recommend a veterinary orthotic device to a friend when owner perceptions of pet quality of life were positive as compared to negative or neutral. Willingness to recommend an experience to a friend is a reflection of satisfaction with the experience. The dependence of owner and pet quality of life should therefore guide therapeutic decisions for patient management and client communication to ensure that the orthosis experience is positive for both patient and owner.

The purpose of this study was to learn how perceptions of accuracy and availability of sources affect how members of the Colorado equine industry seek both everyday information and information during an equine disease outbreak. A survey was distributed by email and social media to members of Colorado-based equine organizations. A total of 256 survey responses were obtained from individuals representing a spectrum of ages and roles in the Colorado equine industry. Survey participants predominantly identified as female (95.3%) and their industry role as a horse owner (41%) or a competitive (25.8%) or pleasure (13.3%) rider. Younger survey participants reported greater (P < .0001) use of social media, and both participant age (P < .015) and information source (P < .0001) affected the perception of resource accuracy. In the event of an equine disease outbreak, industry role was an important factor (P = .003) in the selection of news sources, whereas age was not (P = .19). Many participants (56%) identified disease symptoms/signs to be the most important information to be sought during a disease outbreak and most (69.9%) preferred state or veterinary resources for this information. The identification of why Colorado equine industry members access information from specific sources may guide animal health and extension professionals to tailor their online presence to best meet the communication needs of the Colorado equine industry.

Breeding a bitch with two different sires during a single estrous cycle has the potential to facilitate rapid genetic gain and improve reproductive performance within a canine breeding colony. There is limited data regarding the factors that contribute to the success of dual-sired litters in domestic dogs, and only anecdotal evidence suggesting that these litters rarely produce offspring from more than one sire. The objective of this prospective clinical study was to investigate multiple factors that likely affect the success of dual-sired litters on whelping rate, litter size and parentage ratio. These factors include: timing of artificial insemination (AI), order of sires, number of AI's per cycle, semen type, sperm quality and age of sire and bitch. Data collected over a 10 year period from twenty-nine estrous cycles (28 individual bitches of 10 different breeds) were evaluated after an initial AI with frozen semen from the 'genetically desired' sire and followed up with a second AI with either fresh (n = 9) or frozen (n = 16) semen or natural mating (n = 4) from a different, 'back up', sire. DNA parentage of each pup born was determined by using a primary panel of 288 SNPs. The whelping rate and litter size from previous single sire inseminations per estrous cycle, in the same bitches, (n = 16) over 25 estrous cycles using either fresh (n = 4) or frozen-thawed (n = 21) semen, were analyzed as controls. Of the 29 dual-sired breedings, 26 bitches whelped (89.7%), and 8 litters (30.8%) were of mixed parentage. In the litters of mixed parentage after a dual-sired breeding, a greater proportion of the offspring were from the second sire than the first sire (73.0% and 27.0% respectively; P < 0.05). Interestingly, in litters where all pups were of single sire parentage after a dual-sired breeding, 50.0% of the offspring were by the first sire and 50.0% were by the second sire. For litters of mixed or single paternity produced by dual-sired breeding there was no difference in average litter size. However, on a per estrous cycle basis for each bitch the whelping rate (89.7% v. 76.0%.) and litter size (5.5 ± 2.5 v. 4.0 ± 2.78) of all dual-sired breedings were greater (P < 0.05) compared to previous single-sired breedings (controls) respectively. This study demonstrates that offspring of mixed parentage derived from dual-sired breedings may be achieved. Furthermore, insemination with semen from two different sires may increase the whelping rate and litter size, which is an important consideration when using genetically valuable, or older individuals with potentially reduced fertility.

To more clearly understand the equine gonadotrope response to kisspeptin and gonadotropin releasing hormone (GnRH), peripheral LH and FSH were quantified in diestrous mares after treatment with either equine kisspeptide (eKp-10, 0.5 mg iv), GnRH (25 mu g iv), or a combination thereof every 4 h for 3 days. The following observations were made: 1) a diminished LH and FSH response to eKp-10 and GnRH was observed by Day 3, but was not different by treatment, 2) a decrease in basal LH concentration was observed from Day 1 to Day 3 for the eKp-10, but not the GnRH treated mares, 3) there was no change in basal FSH with either treatment. Additionally, pre-treatment with GnRH antagonist (antide 1.0 mg iv) eliminated any measurable change in LH after eKp-10 (1.0 mg iv) treatment. Both GnRH and kisspeptin are G alpha(q/11) coupled receptors, therefore quantifying the rise in intracellular calcium following treatment with cognate ligand allows simultaneous assessment of receptor activation. Direct stimulation of equine primary pituitary cells with GnRH and/or eKp-10 demonstrates three distinct populations of pituitary cells: one population responded to both eKp-10 and GnRH, a second, independent population, responded to only eKp-10, and a third population responded only to GnRH. These populations were confirmed using co-immunofluorescence of hemipituitaries from mares in diestrus. Although the rise in peripheral LH concentration elicited by eKp-10 is dependent on GnRH, this work suggests that kisspeptin also has a specific and direct effect on the equine gonadotrope, independent of GnRH. (C) 2020 Published by Elsevier Inc.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-haCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches. Summary sentence Unique RNA signatures establish the efficacy of combining adenovirus-mediated gene transfer and flow cytometry to isolate an enriched population of gonadotrope cells from the ovine pituitary gland.

PROBLEM:Current methods to quantify kisspeptin (KP) are limited. To this end, a radioimmunoassay (RIA) specific for KP was developed and validated. We hypothesized that use of a RIA would reveal multiple hypothalamic regions as targets of negative seasonal feedback of estradiol on KP production in sheep.METHOD OF STUDY:Ovariectomized (OVX) ewes bearing a subcutaneous implant of estradiol were euthanized during the breeding season (BS) (n = 4) and non-breeding season (NBS) (n = 3). Coronal sections of preoptic area (POA), anterior hypothalamic area (AHA), and mediobasal hypothalamus (MBH) were collected, as well as the median eminence (ME), cortex, brain stem, and cerebellum. Amounts of KP and gonadotropin-releasing hormone (GnRH) in individual hypothalamic nuclei were quantified by radioimmunoassay.RESULTS:Concentration and content of KP were lower during the NBS than the BS in the MBH (P < 0.01) and POA (P < 0.01). Levels of KP in tissue adjacent to the POA and MBH were much lower, and neither concentration nor content of KP differed between the BS and NBS. Kisspeptin was also detected in the cortex, brain stem, and cerebellum, but concentrations were not affected by season. In addition, concentration and content of GnRH in the POA, AHA, MBH, and ME were similar between seasons.CONCLUSION:Our RIA results indicate that in addition to the MBH, the POA and AHA appear to be involved in the seasonal negative feedback of estradiol on KP expression.

IntroductionUsing case-based learning (CBL) in classrooms is becoming increasing popular on college campuses across the country. Even professional schools such as Harvard Medical School are changing large portions of their curriculum to casebased learning in order to emphasize learning to learn, rather than rote memorizations (Shaw, 2015). Using case studies as a means to solve novel problems allows students to apply their knowledge of the subject in a way that forces thinking beyond what they are able to memorize from notes and textbook. In addition to giving students more experience with the subject matter, case studies bridge the gap between theory and practice and between the academy and the workplace (Barkley, Cross, and Major, 2005). One of the benefits of case based learning from a pedagogical perspective is that solving case studies pushes students into almost every level of Bloom's taxonomy. In addition, this method is learner-centered and involves intense interaction between the participants. CBL focuses on the building of knowledge and process of solving problems, and the group works together to examine the case. The instructor's role is that of a facilitator, and the students collaboratively address problems from a perspective that requires analysis (Queen's University, 2011). Teaching students an approach to solving novel problems will not only help them retain subject-matter information more effectively, but it will also give students an approach by which to solve novel problems in other academic venues as well as real-life scenarios. Novel problems are sometimes so complex that a student's usual approach of simply using recall is not effective.However, as educators we know that there are many learning tools that are implemented with varied success depending on the strength of the facilitator. Case-based learning is no different. In order for case studies to be effective in the classroom, the facilitator must carefully select and prepare cases that lead students to ask themselves more questions and to think critically in a sequence of 4 steps:1. Define the unknown2. What is the timeline?3. What systems are involved?4. What is unique?This 4-step approach was derived in an Anatomy classroom at the university level, but has been tested and proven successful with high school students, undergraduates, and graduate students.What makes a great case study?Effective case studies generally have the following elements:* Main character who has a problem that needs to be solved* A description of the problem in context* Supporting data that will lead students to the relevant questions they need to ask in order to solve the problem (this depends on the context you are giving the students but can range from character testimonials to scientific data)* Includes superfluous information that students learn to recognize as not having relevance or importance.Case studies should be simple enough so that students can successfully solve the problem in the allotted time period, yet complex enough to cover the intended learning objectives. According the Herreid, Schiller, Herreid, and Wright from the National Center for Case Study Teaching in Sciences, (2015), a good case study (a) tells a story, (b) focuses on an interest-arousing issue, (c) is set in the past 5 years, (d) creates empathy with the central characters, (e) includes quotations and dialogue, (f) is relevant to the reader, (g) must have pedagogical utility (i.e., it must serve a teaching function), (h) is conflict provoking, (i) is decision forcing, (j) has generality, and (k) is short. There are many resources available in print as well as online with pre-written case studies across multiple disciplines. However, if an appropriate case study does not already exist, following the above criteria will help you craft a case study to fit your specific discipline.How to Implement Case Studies in a ClassroomOne of the common downfalls of teaching case studies is the teacher's desire to point the students to the answer by asking a set of leading questions. …

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.