Differential CRE Expression in Lhrh-cre and GnRH-cre Alleles and the Impact on Fertility in Otx2-Flox Mice.

There is an increasing trend in studies utilizing cell-specific deletion of genes through conditional gene deletion by CRE recombination. Despite numerous advantages, this strategy also has limitations such as ectopic CRE-expression and germline recombination. Two commonly used gonadotropin-releasing hormone (Gnrh)-driven CRE-expressing mice both target GnRH neurons. However, a direct comparison of the cells targeted and their phenotypic outcome have not yet been presented. To compare where recombination takes place, we crossed the Gnrh-cre and Lhrh-cre lines with the Rosa26-LacZ reporter mouse. Lhrh-cre allowed recombination of the Rosa26-LacZ gene in ∼700 cells, which is comparable to the GnRH neuronal population. Surprisingly, there were > 20 times more LacZ expressing cells in the adult Gnrh-cre:Rosa26-LacZ than the Lhrh-cre:Rosa26-LacZ brain. The greatest differences in targeting of the Gnrh-cre and Lhrh-cre lines were found in the septum, the suprachiasmatic nucleus, and the septohypothalamic area. This difference in cells targeted was present from embryonic day 12. A prior study using the Gnrh-cre to delete the transcription factor Otx2 found fewer GnRH neurons, leading to male and female subfertility. To recapitulate this study, we performed a fertility assay in Otx2:Lhrh-cre mice. We confirmed the requirement for Otx2 in GnRH neuron development, fertility and correct gonadotropin hormone release in Otx2:Lhrh-cre males, but the subfertility was more modest than in Otx2:Gnrh-cre and absent in female Otx2:Lhrh-cre. This suggests that ectopic expression of Gnrh-cre contributes to the reproductive phenotype observed. Finally, the Cre alleles caused germline recombination of the flox allele when transmitted from either parent, generating embryonic lethal knock-out offspring, producing smaller live litters.

Characterization of the recombinant (R)- and (S)-hydroxypropyl-coenzyme M dehydrogenases: A case study to augment the teaching of enzyme kinetics and stereoselectivity.

A homologous pair of stereospecific NAD-dependent enzymes, (R)- and (S)-hydroxypropyl-coenzyme M dehydrogenase, are part of a bacterial pathway of short-chain alkene and epoxide metabolism. Their discovery and study, which spans multiple publications over more than a decade, is a data rich story that combines both classical and contemporary experimental biochemistry. A subset of the data for characterization of the recombinant enzymes was used as a case study to augment the teaching of enzyme kinetics and stereoselectivity in an undergraduate biochemistry course at California State University-Chico. © 2018 International Union of Biochemistry and Molecular Biology, 47(2): 124-132, 2019.

Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates.

Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2).  This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions.  Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations.  Samples at equilibrium were infused directly into the mass spectrometer under native conditions.  For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.

Virtual protein purification: A simple exercise to introduce ph as a parameter that effects ion exchange chromatography.

This article describes a simple exercise using a free, easy-to-use, established online program. The exercise helps to reinforce protein purification concepts and introduces undergraduates to pH as a parameter that affects anion-exchange chromatography. The exercise was tested with biochemistry majors at California State University-Chico. Given the versatility of the program, this work is also a model for instructors that wish to develop their own exercise to help teach other protein purification techniques. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):91-97, 2018.

Bmal1 Is Required for Normal Reproductive Behaviors in Male Mice.

Circadian rhythms synchronize physiological processes with the light-dark cycle and are regulated by a hierarchical system initiated in the suprachiasmatic nucleus, a hypothalamic region that receives direct photic input. The suprachiasmatic nucleus then entrains additional oscillators in the periphery. Circadian rhythms are maintained by a molecular transcriptional feedback loop, of which brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a key member. Disruption of circadian rhythms by deletion of the BMAL1 gene (Bmal1 knockout [KO]) induces a variety of disease states, including infertility in males, due to unidentified mechanisms. We find that, despite normal sperm function, Bmal1 KO males fail to mate with receptive females, indicating a behavioral defect. Mating is dependent on pheromone detection, as are several other behaviors. We determined that Bmal1 KO males also fail to display aggression and avoidance of predator scent, despite intact main olfactory function. Moreover, the vomeronasal organ, a specialized pheromone-responsive organ, was also functionally intact, as determined by calcium imaging in response to urine pheromone stimulus. However, neural circuit tracing using c-FOS activation revealed that, although Bmal1 KO males displayed appropriate activation in the olfactory bulb and accessory olfactory bulb, the bed nucleus of the stria terminalis and the medial preoptic area (areas responsible for integration of copulatory behaviors) failed to activate highly in response to the female scent. This indicates that neural signaling in select behavioral centers is impaired in the absence of BMAL1, likely underlying Bmal1 KO male copulatory defects, demonstrating the importance of the BMAL1 protein in the maintenance of neural circuits that drive pheromone-mediated mating behaviors.

Gene dosage of Otx2 is important for fertility in male mice.

Together, the hypothalamus, pituitary and gonads direct the development and regulation of reproductive function in mammals. Gonadotropin-releasing hormone (GnRH) expression is limited to ∼800 neurons that originate in the olfactory placode then migrate to the hypothalamus. Coordination of the hypothalamic-pituitary-gonadal (HPG) axis is dependent upon correct neuronal migration of GnRH neurons into the hypothalamus followed by proper synthesis and pulsatile secretion of GnRH. Defects in any one of these processes causes infertility. Otx2, the vertebrate homologue of Drosophila orthodenticle, is a transcription factor that has been shown to be critical for normal brain and eye development and is expressed in both the developing GnRH neurons and the pituitary, suggesting that this gene may play a critical role in development of the HPG axis. As Otx2-null mice are embryonic lethal, we have analyzed the reproductive capacity of heterozygous Otx2 mice to determine the contribution of Otx2 gene dosage to normal HPG axis function. Our data reveal that correct dosage of Otx2 is critical for normal fertility as loss of one allele of Otx2 leads to a discernible reproductive phenotype in male mice due to disruption of the migration of GnRH neurons during development.

Aberrant development of the suprachiasmatic nucleus and circadian rhythms in mice lacking the homeodomain protein Six6.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and ß-galactosidase, we demonstrated the lack of these SCN markers in all Six6-null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN.

Hypothalamic dysregulation and infertility in mice lacking the homeodomain protein Six6.

The hypothalamus, pituitary, and gonads coordinate to direct the development and regulation of reproductive function in mammals. Control of the hypothalamic-pituitary-gonadal axis is dependent on correct migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus, followed by proper synthesis and pulsatile secretion of GnRH, functions absent in patients with hypogonadal hypogonadism. In this study, we identify sine oculis-related homeobox 6 (Six6) as a novel factor necessary for proper targeting of GnRH expression to the limited population of GnRH neurons within the adult mouse hypothalamus and demonstrate that it is required for proper reproductive function in both male and female mice. Female Six6-null mice exhibit a striking decrease in fertility, failing to progress through the estrous cycle normally, show any signs of successful ovulation, or produce litters. Although basal gonadotropin production in these mice is relatively normal, analysis of GnRH expression reveals a dramatic decrease in total GnRH neuron numbers. We show that expression of Six6 is dramatically increased during GnRH neuronal maturation and that overexpression of Six6 induces GnRH transcription in neuronal cells. Finally, we demonstrate that this induction in GnRH expression is mediated via binding of Six6 to evolutionarily conserved ATTA sites located within the GnRH proximal promoter. Together, these data indicate that Six6 plays an important role in the regulation of GnRH expression and hypothalamic control of fertility.

Shotgun proteomics of Xanthobacter autotrophicus Py2 reveals proteins specific to growth on propylene.

Coenzyme M (CoM, 2-mercaptoethanesulfonate), once thought to be exclusively produced by methanogens, is now known to be the central cofactor in the metabolism of short-chain alkenes by a variety of aerobic bacteria. There is little evidence to suggest how, and under what conditions, CoM is biosynthesized by these organisms. A shotgun proteomics approach was used to investigate CoM-dependent propylene metabolism in the Gram-negative bacterium Xanthobacter autotrophicus Py2. Cells were grown on either glucose or propylene, and the soluble proteomes were analyzed. An average of 395 proteins was identified from glucose-grown replicates, with an average of 419 identified from propylene-grown replicates. A number of linear megaplasmid (pXAUT01)-encoded proteins were found to be specifically produced by growth on propylene. These included all known to be crucial to propylene metabolism, in addition to an aldehyde dehydrogenase, a DNA-binding protein, and five putative CoM biosynthetic enzymes. This work has provided fresh insight into bacterial alkene metabolism and has generated new targets for future studies in X. autotrophicus Py2 and related CoM-dependent alkene-oxidizing bacteria.

Mechanism of inhibition of aliphatic epoxide carboxylation by the coenzyme M analog 2-bromoethanesulfonate.

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys(82)) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys(87) was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys(82). These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.