Timothy G. Rozell

Development of recombinant ovalbumin-luteinizing hormone releasing hormone as a potential sterilization vaccine

The objective was to develop an immunogenic chimeric ovalbumin-LHRH (ova-LHRH) molecule using genetic engineering. Hybrid ova-LHRH genes with either four or seven LHRH inserts were constructed by cassette mutagenesis and oligonucleotide mismatch mutagenesis. Recombinant ova-LHRH proteins were over-expressed in E. coli strain BL21 (DE3) using a pET expression system, which expresses a target protein with a C-terminal His-Tag. The C-terminal His-Tag allows purification by metal chelation chromatography. The antigenicity and biological effects of these recombinant proteins were tested in mice. In experiment 1, 17 female 7 wk old BALB/c mice were randomly divided into three groups. Six mice were injected with 50 microg of the recombinant ovalbumin (ova) protein. Five mice were injected with 50 microg of the recombinant protein with four LHRH inserts (ova-LHRH-7). Six mice were injected with 50 microg of the recombinant protein with seven LHRH inserts (ova-LHRH-7). One primary immunization using Freunds complete adjuvant was followed by one booster using incomplete adjuvant. Mice were killed 2 wk after the booster, blood collected, and the reproductive tract removed and weighed. Only ova-LHRH-7 decreased (P < 0.01) uterine-ovarian weight (89+/-11 mg) vs control (138+/-6 mg) and ova-LHRH-4 (126+/-16 mg). The genetically engineered molecule with seven LHRH inserts induced LHRH antibody titers which were significantly correlated (r = -0.79) with biological response. In experiment 2, the recombinant ova-LHRH-7 was evaluated at two doses with the adjuvants Zmax and Immumax. Seventy female 6-8 wk old BALB/c mice were randomly divided into seven groups of 10 mice each. Anti-LHRH titers were detected in all of the ova-LHRH-7 immunized mice. Significant decreases were shown in uterine-ovarian weight of the mice by the immunization with 30 microg of ova-LHRH-7 and Zmax (P < 0.005) or 10 microg of ova-LHRH-7 with Immumax (P < 0.025). These data show that the recombinant ova-LHRH-7 protein could have potential as an effective sterilization vaccine.

Inhibition of Vascular Endothelial Growth Factor Receptor Signal Transduction Blocks Follicle Progression but Does Not Necessarily Disrupt Vascular Development in Perinatal Rat Ovaries

We hypothesized that vascular endothelial growth factor A (VEGFA) angiogenic isoforms and their receptors, FLT1 and KDR, regulate follicular progression in the perinatal rat ovary. Each VEGFA angiogenic isoform has unique functions (based on its exons) that affect diffusibility, cell migration, branching, and development of large vessels. The Vegfa angiogenic isoforms (Vegfa_120, Vegfa_164, and Vegfa_188) were detected in developing rat ovaries, and quantitative RT-PCR determined that Vegfa_120 and Vegfa_164 mRNA was more abundant after birth, while Vegfa_188 mRNA was highest at Embryonic Day 16. VEGFA and its receptors were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. To determine the role of VEGFA in developing ovaries, Postnatal Day 3/4 rat ovaries were cultured with 8 μM VEGFR-TKI, a tyrosine kinase inhibitor that blocks FLT1 and KDR. Ovaries treated with VEGFR-TKI had vascular development reduced by 94% (P < 0.0001), with more primordial follicles (stage 0), fewer early primary, transitional, and secondary follicles (stages 1, 3, and 4, respectively), and greater total follicle numbers compared with control ovaries (P < 0.005). V1, an inhibitor specific for KDR, was utilized to determine the effects of only KDR inhibition. Treatment with 30 μM V1 had no effect on vascular density; however, treated ovaries had fewer early primary, transitional, and secondary follicles and more primary follicles (stage 2) compared with control ovaries (P < 0.05). We conclude that VEGFA may be involved in primordial follicle activation and in follicle maturation and survival, which are regulated through vascular-dependent and vascular-independent mechanisms.

The balance of proangiogenic and antiangiogenic VEGFA isoforms regulate follicle development

Vascular endothelial growth factor A (VEGFA) has been extensively studied because of its role in follicular development and is a principal angiogenic factor essential for angiogenesis. Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, VEGFA might influence follicle growth via the regulation of angiogenesis. However, VEGFA might also influence follicular development through nonangiogenic mechanisms, since its expression has been localized in nonvascular follicles and cells. Alternative mRNA splicing of eight exons from the VEGFA gene results in the formation of various VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms (VEGFA_XXX) are encoded by exon 8a, whereas a sister set of isoforms (VEGFA_XXXB) with antiangiogenic properties is encoded by exon 8b. The antiangiogenic VEGFA_XXXB isoforms comprise the majority of VEGFA expressed in most tissues, whereas expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. Although proangiogenic and antiangiogenic isoforms can now be distinguished from one another, many studies evaluating VEGFA in ovarian and follicular development up to now have not differentiated proangiogenic VEGFA from antiangiogenic VEGFA. Experiments from our laboratory indicate that proangiogenic VEGFA promotes follicle recruitment and early follicular development and antiangiogenic VEGFA inhibits these processes. The balance of proangiogenic versus antiangiognic VEGFA isoforms is thus of importance during follicle development. Further studies are warranted to elucidate the way that this balance regulates follicular formation and progression.

A constructivist approach to e-text design for use in undergraduate physiology courses

Electronic textbooks, or e-texts, will have an increasingly important role in college science courses within the next few years due to the rising costs of traditional texts and the increasing availability of software allowing instructors to create their own e-text. However, few guidelines exist in the literature to aid instructors in the development and design specifically of e-texts using sound learning theories; this is especially true for undergraduate physiology e-texts. In this article, we describe why constructivism is a very important educational theory for e-text design and how it may be applied in e-text development by instructors. We also provide examples of two undergraduate physiology e-texts that were designed in accordance with this educational theory but for learners of quite different backgrounds and prior knowledge levels.

Effects of vaccinating beef dams precalving and calves preweaning with a Pasteurella haemolytica vaccine

Our objective was to determine if vaccinating dams precalving and calves preweaning for Pasteurella haemolytica could effect serum antibody titers in dams, and the preand post-weaning health and performance of their calves. Vaccination increased serum antibody titers in multiparous cows, but not first-calf heifers. Precalving vaccination had minimal effects on mortality and morbidity of calves before or after weaning. Subsequent steer feedlot gains were unaffected by precalving and preweaning vaccinations and carcasses were not affected. However, heifers’ weight gains were greater from weaning to one year of age when reared by vaccinated dams.

Cognitive flexibility and undergraduate physiology students: increasing advanced knowledge acquisition within an ill-structured domain

Cognitive flexibility is defined as the ability to assimilate previously learned information and concepts to generate novel solutions to new problems. This skill is crucial for success within ill-structured domains such as biology, physiology, and medicine, where many concepts are simultaneously required for understanding a complex problem, yet the problem consists of patterns or combinations of concepts that are not consistently used or needed across all examples. To succeed within ill-structured domains, a student must possess a certain level of cognitive flexibility: rigid thought processes and prepackaged informational retrieval schemes relying on rote memorization will not suffice. In this study, we assessed the cognitive flexibility of undergraduate physiology students using a validated instrument entitled Students Approaches to Learning (SAL). The SAL evaluates how deeply and in what way information is processed, as well as the investment of time and mental energy that a student is willing to expend by measuring constructs such as elaboration and memorization. Our results indicate that students who rely primarily on memorization when learning new information have a smaller knowledge base about physiological concepts, as measured by a prior knowledge assessment and unit exams. However, students who rely primarily on elaboration when learning new information have a more well-developed knowledge base about physiological concepts, which is displayed by higher scores on a prior knowledge assessment and increased performance on unit exams. Thus students with increased elaboration skills possibly possess a higher level of cognitive flexibility and are more likely to succeed within ill-structured domains.

Differences in serum immunoglobulin g1 and total protein concentrations in neonatal calves on days 1, 5, and 10

Immunoglobulin G1 (IgG1) serum concentrations are used to evaluate passive transfer of immunity in neonatal calves. Total serum proteins also can be measured to evaluate calf health. If IgG1 and total serum protein concentrations change with age, it becomes imperative to compare samples only from a narrow time period. Otherwise, differences might be due to age and not immune status. To help define this time period, blood was drawn from 10 beef calves when they were 1, 5, and 10 days of age. Serum samples were analyzed for IgG1 and total protein concentrations. Total protein concentrations decreased from days 1 to 5 (P<.05) or days 1 to 10 (P<.05), but not from days 5 to 10 (P=.46). IgG1 concentrations declined from days 1 to 10 (P<.05), but values from days 1 to 5 were similar (P=.17). Thus, it is important to collect serum on day 1 to guarantee correct results when evaluating IgG1 and total proteins collectively. However, if IgG1 alone is evaluated, serum can be collected between days 1 and 5.

Increasing pregnancy rates at first service in dairy cows exposed to high ambient temperatures before and after calving

Cows exposed to heat stress before or after calving or both are prone to reduced fertility because of reduced expression of estrus and less embryonic survival if pregnant. Cows calving on three dairy farms during the summer of 1998 were studied. First inseminations were programmed to occur between 50 and 70 days in milk using the Ovsynch protocol, which included a timed artificial insemination. Control cows were treated similarly but did not receive the second injection of gonadotropin-releasing hormone and were inseminated only after estrus was detected (Select Synch). The Ovsynch protocol increased pregnancy rates from 17.6 to 31.3%, because AI submission rates were 100% and conception rates were not different from those of control (Select Synch) cows.

Site of semen deposition and fertility in lactating beef cows synchronized with GnRH and PGF2a

Our objective was to determine the effect of site of semen deposition on pregnancy rate in beef cows inseminated at a fixed time or after observed estrus. Cows were synchronized with a combination of gonadotropin-releasing hormone (GnRH) prostaglandin-F α (PGF). GnRH was injected 7 days before PGF (day 0; first of breeding season). The trial was conducted at two locations, one in Kansas (147 cows) and one in Colorado (313 cows). At each location, cows were assigned to be inseminated after observed estrus (ESTRUS-AI) or at a fixed time (TIMED-AI). Within these two groups, cows either were inseminated in the uterine body (BODY-bred) or in both uterine horns (HORN-bred). Cows in the ESTRUS-AI group were observed for estrus each morning and evening until day 5 afterPGF and then inseminated 12 hr after first detected estrus. Cows in the TIMED-AI group received a second dose of GnRH on day 2 and were inseminated at that time (48 to 56 hr after PGF). Heat response, AI conception rate, and pregnancy rate were analyzed for BODY-bred and HORN-bred cows within each treatment at each location. No differences in these variables occurred between locations, so the results were combined. Within the ESTRUS-AI group, neither conception rate (70% vs. 73%) nor pregnancy rate (39% vs. 40%) was different between BODY-bred and HORN-bred cows respectively. Pregnancy rate within the TIMED-AI group tended (P=.09) to be greater for BODY-bred (53%) compared to HORN-bred (42%) cows. When BODY-bred and HORN-bred treatments were combined, the pregnancy rate of TIMED-AI cows (48%) tended (P=.07) to be greater than that of ESTRUS-AI cows (39%). Timed-insemination resulted in a greater pregnancy rate than inseminating cows according to estrus. No advantage was seen in conception rates when semen was deposited in the uterine horns compared to the uterine body.