Domestic Cat Model Research Articles

Proteomic analysis of germinal vesicles in the domestic cat model reveals candidate nuclear proteins involved in oocyte competence acquisition.

Do nuclear proteins in the germinal vesicle (GV) contribute to oocyte competence acquisition during folliculogenesis? Proteomic analysis of GVs identified candidate proteins for oocyte competence acquisition, including a key RNA processing protein-heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1). The domestic cat GV, which is physiologically similar to the human GV, gains the intrinsic ability to resume meiosis and support early embryo development during the pre-antral-to-antral follicle transition. However, little is known about nuclear proteins that contribute to this developmental process. GVs were enriched from pre-antral (incompetent) and antral (competent) follicles from 802 cat ovaries. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in GVs from the two follicular categories. Two biological replicates (from independent pools of ovaries) of pre-antral versus antral samples were labeled by tandem mass tags and then assessed by liquid chromatography-tandem mass spectrometry. Proteomic data were analyzed according to gene ontology and a protein-protein interaction network. Immunofluorescent staining and protein inhibition assays were used for validation. A total of 174 nuclear proteins was identified, with 54 being up-regulated and 22 down-regulated (≥1.5-fold) after antrum formation. Functional protein analysis through gene ontology over-representation tests revealed that changes in molecular network within the GVs during this transitional phase were related to chromatin reorganization, gene transcription, and maternal RNA processing and storage. Protein inhibition assays verified that hnRNPA2B1, a key nuclear protein identified, was required for oocyte meiotic maturation and subsequent blastocyst formation. Data are available via ProteomeXchange with identifier PXD007211. Proteins identified by proteomic comparison may (i) be involved in processes other than competence acquisition during the pre-antral-to-antral transition or (ii) be co-expressed in other macrostructures besides the GV. Expressional and functional validations should be performed for candidate proteins before downstream application. Collective results generated a blueprint to better understand the molecular mechanisms involved in GV competence acquisition and identified potential nuclear competence markers for human fertility preservation. Funded by the National Center for Research Resources (R01 RR026064), a component of the National Institutes of Health (NIH) and currently by the Office of Research Infrastructure Programs/Office of the Director (R01 OD010948). The authors declare that there is no conflict of interest.

Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model

Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function.

Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model

Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the ‘Non-cultured control’. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.

The nuclear and developmental competence of cumulus-oocyte complexes is enhanced by three-dimensional coculture with conspecific denuded oocytes during in vitro maturation in the domestic cat model.

The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.

Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation. Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200nM) for 10 or 120min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200nM FCCP for 120min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density. Ovarian tissues incubated with 200nM FCCP for 120min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8%) similar to the control group (71.8 ± 11.7%; P > 0.05). After 2days of in vitro culture, percentage of viable follicles (78.8 ± 8.9%) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0%) with percentages of morphologically normal follicles (57.6 ± 17.3%) not different from the fresh tissue (70.2 ± 7.1%; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. Pre-exposure to 200nM FCCP during 120min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

The preservation of vital functions in cat ovarian tissues during vitrification depends more on the temperature of the cryoprotectant exposure than on the sucrose supplementation

The objective of this study was to better characterize the impact of cryoprotectant exposure (temperature and sucrose supplementation) on the health and function of preantral follicles in ovarian tissues during vitrification using the domestic cat model. Ovarian cortical pieces from peri-pubertal individuals were exposed to cryoprotectants at 4 °C or room temperature and supplemented with 0 or 0.5 M of sucrose, followed by vitrification. After rapid warming, cortical pieces were cultured in vitro and assessed for normal follicular morphology, viability and resumption of transcriptional activities for up to 7 days. Throughout the culture period, follicular morphology (up to 67.5% normal follicles) and global RNA transcription (up to 50.9% follicles with transcriptional activity) in warmed tissues were improved by cryoprotectant exposure at 4 °C compared to room temperature, but viability (up to 84.6% viable follicles) did not seem to be affected by exposure temperature. Sucrose supplementation did not have a consistent effect as it increased RNA transcription but decreased normal follicular morphology. For the first time, the study demonstrated that the preservation of critical tissue functions, such as the transcriptional activities, highly depends on the temperature of the cryoprotectant exposure and not necessarily on the presence of sucrose.

Incidence of methylated histones H3K4 and H3K79 in cat germinal vesicles is regulated by specific nuclear factors at the acquisition of developmental competence during the folliculogenesis.

This study aims to characterize the regulations of histone methylations, key epigenetic markers of oocyte competence, in germinal vesicle (GV) from different follicles (preantral, early, small, or large antral stage) using the domestic cat model. In Experiment 1, the incidence of H3K4me3 or H3K79me2 was determined in GVs from the diverse follicle stages directly or after exposure to (1) a methyltransferase inhibitor, (2) sonication to fracture the cytoplasmic membranes and wash away the cytoplasmic content, or (3) methyltransferase inhibitor followed by sonication. In Experiment 2, the presence and maintenance of nuclear methyltransferases SMYD3 and DOT1L (regulating H3K4me3 and H3K79me2, respectively) was characterized in separate GV stages before and after sonication. Functionality of GVs from the various follicle stages (with or without transient isolation from the cytoplasm) then was assessed in Experiment 3 by transfer into recipient competent oocytes. The incidence of histones H3K4me3 and H3K79me2 within the GV were influenced by the cytoplasmic environment at all stages except at the transition to the early antral stage where nuclear regulating factors appeared to be mainly involved. The methyltransferase SMYD3 and DOT1L also appeared tightly bound to the nucleus at that transition. Interestingly, oocytes reconstructed with a GV isolated from the cytoplasm for a prolonged period had the capacity to form an embryo after fertilization which proved that communication between the donor GV and the host cytoplasm (likely including the regulation of epigenetic factors) could be restored. Histone methylation apparently becomes regulated by specific nuclear factors at the acquisition of competence during the folliculogenesis and does not seem to be disrupted by prolonged isolation from the surrounding cytoplasm.

Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model.

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased (p<0.05) from 1.7 to 0.1 gH2O/gDW over the first 30 min, but did not decrease further (p>0.05) after 40 min. Chromatin configuration was unaffected (p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered (p>0.05) from 0 to 30 min of treatment (range, 6.1%-12%), but increased (p<0.05) to 32.5% after 40 min. Next, the influence of storage at two different supra-zero temperatures after 30 min of drying was investigated. Oocyte-loaded, microwave-treated filters were individually sealed in Dri-Shield moisture barrier bags and stored at 4°C or ambient temperature for 0 to 8 weeks. Moisture contents gradually decreased (p<0.05) from 0.12 to 0.10 gH2O/gDW after 8 weeks of storage at 4°C or ambient temperature. The percentage of GVs with DNA fragmentation more than doubled (p<0.05) from 0 (14.3%) to 2 days (30.0%-33.0%), but remained stable (p>0.05) thereafter (1 through 4 weeks, 25.0%-35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature.

C-Type natriuretic peptide maintains domestic cat oocytes in meiotic arrest.

Recent studies have shown that C-type natriuretic peptide (CNP; encoded by the natriuretic peptide C (NPPC) gene) plays an essential role in maintaining meiotic arrest of mouse and porcine oocytes. However, whether CNP inhibits feline meiotic resumption is not known. In the present study we used a domestic cat model to explore the role played by CNP in feline oocyte meiotic resumption. We determined mRNA expression of genes encoding CNP and its cognate receptor natriuretic peptide receptor 2 (NPR2) in antral follicles. NPPC mRNA was primarily expressed in mural granulosa cells, whereas NPR2 mRNA was predominantly expressed in cumulus cells. Following in vitro culture for 24h, 100nM CNP increased cGMP levels, and maintained meiotic arrest of oocytes associated with cumulus cells. When the duration of in vitro culture increased from 24h to 36h, the ability of CNP to maintain meiotic arrest decreased, and this was accompanied by a decrease in the steady state levels of NPR2 mRNA in cumulus cells. In addition, CNP decreased the rate of degeneration of oocytes. These results indicate that CNP is required to maintain meiotic arrest and prevent degeneration in domestic cat oocytes.

086 Optimal preservation of DNA Integrity in cat germinal vesicles after microwave-assisted dehydration and supra-zero temperature storage

The germinal vesicle (GV) is an alternative target to the whole oocyte for exploring new genome preservation approaches at supra-zero temperatures. Using the domestic cat model, we have demonstrated that the GV can survive simple air-drying and then be hydrated to reconstitute a viable oocyte (Graves-Herring et al., 2013). However, this method can lead to variable DNA damage due to uncontrolled water loss during the dehydration and storage processes. The objective of the present study was to better understand the role of water content during these critical steps on GV DNA integrity. Intraovarian immature oocytes collected from adult cat ovaries were denuded and permeabilized before exposure to 1.5 M trehalose for 10 min. Oocytes were transferred onto conjugate-release filters and dehydrated with a CEM SAM 255 microwave system. In Experiment 1, samples (n = 337 total oocytes, 4–5 replicates) were dehydrated at 20% power for 0, 5, 10, 15, 20, 25, 30 or 40 min. Moisture contents were assessed immediately after microwaving using volumetric Karl Fischer titration. Oocytes were rehydrated to assess chromatin configuration (Hoechst staining) and DNA fragmentation (TUNEL assay). Moisture contents progressively decreased (P 0.05) after 40 min. Chromatin configuration and proportions of degenerated oocytes (range, 2.2–9.1%) were unaffected (P > 0.05) over time. The incidence of GVs with DNA fragmentation was unaltered (P > 0.05) from 0 to 30 min of treatment (range, 6.1–12%), but increased (P 0.05) thereafter (1 through 4 weeks, 25.0–35.0%). This study characterizes, for the first time, the metrics and kinetics of moisture removal, degeneration and DNA damage related to GV desiccation as an alternative approach for preserving the maternal genome at cool or ambient temperature. Findings demonstrate the feasibility for successful microwave dehydration of the mammalian GV to reach an equilibrium moisture content that is non-lethal. Most DNA disruption occurs within the first 2 days of storage at supra-zero temperatures and more than two-thirds of GVs retain intact DNA regardless of further moisture loss during storage. Source of funding: National Institutes of Health. Conflict of interest: None declared. comizzolip@si.edu

Fearing the feline: domestic cats reduce avian fecundity through trait‐mediated indirect effects that increase nest predation by other species

Summary Urban areas contain high densities of non‐native species, which in the UK include the domestic cat Felis catus (Linnaeus 1758) and the grey squirrel Sciurus carolinensis (Gmelin 1788). The direct predation effects of domestic cats on prey populations attract intense debate, and such influences of the nest‐predatory grey squirrel are receiving increasing attention. In contrast, theory predicts that sublethal and indirect effects are more important, but empirical evidence is currently lacking. We conducted controlled model presentation experiments at active urban blackbird Turdus merula (Linnaeus 1758) nests to provide the first empirical evidence that quantifies the potential sublethal and indirect effects of predators (domestic cat and grey squirrel) on avian reproductive success. Domestic cat models reduced subsequent parental provisioning rates, a strong indicator of sublethal effects, by one‐third relative to a nonpredatory rabbit Oryctolagus cuniculus (Linnaeus 1758) control. There was no compensatory increase in food load size. Previous experiments demonstrate that this magnitude of reduced food delivery will reduce nestling growth rates by c. 40%. The grey squirrel model induced similar but weaker effects. Following the brief presence of the domestic cat model, subsequent daily nest predation rates, chiefly by corvids, increased by an order of magnitude relative to the squirrel and rabbit models. The intensity of parental nest defence elicited in response to model presentations predicts the probability of such third‐party predator predation events, and the domestic cat model generated significant increases in nest defence behaviour. Synthesis and applications. The brief presence of a domestic cat at avian nest sites reduces subsequent provisioning rates and induces lethal trait‐mediated indirect effects. We provide the first robust evidence for these latter effects in any avian predator–prey system, although they are likely to be common in many avian assemblages with high predator densities. It is imperative that future assessments of the impact of predatory species on avian prey species take lethal trait‐mediated indirect effects into account, as so doing will prevent biased estimates of predator effects and facilitate the design of more effective control strategies. Full mitigation of the sublethal and indirect effects of domestic cats would problematically require permanent indoor housing.

Feline Gonads Exhibit Tissue Specific Alternative Splicing of Oestrogen Receptor Alpha (ESR1)

Male felids frequently show teratospermia. At least in the domestic cat model, teratospermia is accompanied by impaired regulation of testicular apoptosis. We hypothesize that this phenomenon is caused by dysregulations in oestrogen signalling pathways. Both classical oestrogen receptors (ESR1 and 2) are expressed in species and/or tissue-specific manners and display different variants, inter alia, caused by alternative splicing. In vitro studies showed that exon deleted transcripts are translated into proteins and that some of the variants modify the effects of the full-length ERs. It has been proposed that some of the functional and morphological dysregulations, for example, during spermatogenesis, could directly derive from this phenomenon. In the present basic study, we investigated the expression pattern of ESR1 splicing variants in the gonads of domestic cats. Testicular, epididymal as well as ovarian tissue samples were collected from routine castrations. ESR1 variants were detected by means of RT-PCR using primers spanning one to three exons. We detected the variants Δ4 and Δ7 in all tissue samples investigated. Additionally, the testicular parenchyma expressed the variant Δ6 and double exon deletions of ESR1 (Δ4/6 and Δ6/7). Using an antiserum recognizing all previously identified ESR1 splicing variants, we revealed ESR1 proteins being expressed in nearly all cells of the testicular and ovarian parenchyma. ESR1 Δ6 protein, however, detected by an antiserum specifically raised against the Δ6 variant, was predominantly located in Sertoli cells. As the exon deletion variants are significantly expressed and show a distinct expression pattern, they could specifically modulate the cellular responsiveness to hormonal stimuli within the gonads.

The competence of germinal vesicle oocytes is unrelated to nuclear chromatin configuration and strictly depends on cytoplasmic quantity and quality in the cat model

Chromatin configuration of the germinal vesicle (GV) and quality of the cytoplasm are critical factors in achieving oocyte meiotic and developmental capacity during folliculogenesis. Besides gaining new insights into the timing and cellular mechanisms associated with the acquisition and regulation of GV oocyte competence, the domestic cat model was used to examine (i) the relation between GV chromatin configuration and oocyte functionality during folliculogenesis and (ii) the role of the cytoplasmic environment on the GV competence and stability. Structural and functional properties of GV oocytes were characterized after isolation from different follicle stages of non-stimulated cat ovaries. GV transfers, artificial chromatin compaction and oocyte vitrification were used to demonstrate the respective roles of GV and cytoplasm on the oocyte functionality. GVs acquired the intrinsic capability to resume meiosis during the pre-antral follicle stage, whereas the capacity to support embryo development occurred while the antrum started to form. Chromatin configuration of the GV did not undergo extensive modification during the acquisition of competence or during the arrest of transcriptional activity at the large antral follicle stage. However, the quality and quantity of the cytoplasm regulated and enhanced GV functionality. This finding also held for GVs transferred from incompetent or subpar oocytes into the cytoplasm of good quality oocytes or when chromatin was artificially modified or vitrified. The cat model provides a new insight into GV oocyte structure and function during folliculogenesis while challenging current concepts about oocyte quality criteria based on the GV morphology. This suggests alternative evaluative approaches for oocytes from other species too, including humans. Cat GVs also appear competent at an early follicle stage and are resilient to perturbations which designate this organelle as an attractive target for developing novel fertility preservation tactics.

An ~140-kb deletion associated with feline spinal muscular atrophy implies an essentialLIX1function for motor neuron survival

The leading genetic cause of infant mortality is spinal muscular atrophy (SMA), a clinically and genetically heterogeneous group of disorders. Previously we described a domestic cat model of autosomal recessive, juvenile-onset SMA similar to human SMA type III. Here we report results of a whole-genome scan for linkage in the feline SMA pedigree using recently developed species-specific and comparative mapping resources. We identified a novel SMA gene candidate, LIX1, in an approximately140-kb deletion on feline chromosome A1q in a region of conserved synteny to human chromosome 5q15. Though LIX1 function is unknown, the predicted secondary structure is compatible with a role in RNA metabolism. LIX1 expression is largely restricted to the central nervous system, primarily in spinal motor neurons, thus offering explanation of the tissue restriction of pathology in feline SMA. An exon sequence screen of 25 human SMA cases, not otherwise explicable by mutations at the SMN1 locus, failed to identify comparable LIX1 mutations. Nonetheless, a LIX1-associated etiology in feline SMA implicates a previously undetected mechanism of motor neuron maintenance and mandates consideration of LIX1 as a candidate gene in human SMA when SMN1 mutations are not found.

Long-term, targeted genetic modification of the aqueous humor outflow tract coupled with noninvasive imaging of gene expression in vivo.

To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings. A domestic cat model was developed in which long-term in vivo studies were performed. After dose-response studies in primary human TM cells, 19 cats received anterior chamber (AC) injections of stepped doses (10(6)-10(8) transduction units) of lentiviral vectors encoding different marker transgenes (beta-galactosidase, Aequorea victoria green fluorescent protein [GFP], or Renilla reniformis GFP). Animals were monitored serially for transgene expression and IOP. High-grade, stable transgene expression in the TM was achieved and monitored noninvasively over time in living animals. Extensive expression resulted after a single transcorneal injection, persisted for at least 10 months (time of death in the present studies), and was targeted to the TM. The initial IOP did not differ significantly from the IOP at the end of the study (P = 0.4). Aequorea GFP was superior to Renilla GFP. Vectors were effective enough to cause GFP-specific overexpression cytotoxicity at the highest dose, which was solved by dose reduction. High-grade transgene expression in this large-animal model persisted stably for at least 10 months after a single transcorneal lentiviral vector injection, was highly targeted, and could be monitored serially and noninvasively in living animals. These studies provide a basis for developing realistic disease models and administering glaucoma gene therapy.

Preclinical Overview of WHI-07, A Novel Nucleoside Analog-based Dual- Function Microbicide

The current pandemic of sexually transmitted HIV / AIDS has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. WHI-07 is a novel 5-bromo 6-methoxy aryl phosphoramidate derivative of 3-azido-3-deoxythymidine (zidovudine, ZDV / AZT) with potent anti-HIV and spermicidal activities. WHI-07 was rationally designed to bypass the thymidine kinase dependency of ZDV activation in genital tract secretions. WHI-07 was formulated via a nontoxic gel-microemulsion for intravaginal use as a potential candidate anti-HIV spermicide. The in vivo efficacy as well as safety studies of WHI-07 included: (i) vaginal as well as rectal microbicide efficacy studies in the feline immunodeficiency virus (FIV) / domestic cat model for AIDS; (ii) in vivo retention of anti-HIV activity in the nonhuman primate; (iii) vaginal contraceptive efficacy studies in rabbits; (iv) single- and/ or multiple-dose toxicity studies in mice, rabbits, cats as well as monkeys; (v) mucosal, reproductive, and developmental toxicity studies in mice and rabbits; (vi) cytotoxicity, mutagenicity, and genotoxicy tests using human and yeast cells; and (vii) long-term carcinogenicity studies in mice. WHI-07 which exhibited nanomolar in vitro anti-HIV IC50 and low micromolar spermicidal EC50 values was significantly less active against genital epithelial cells and did not induce primary DNA damage in human and yeast cells. WHI-07 retained full activity in TK-deficient cells, the main carriers of HIV in semen. Both vaginal as well as rectal instillation of WHI-07 gel-microemulsion, either alone or in combination with a vanadocene dithiocarbamate, effectively protected domestic cats from a subsequent vaginal or rectal exposure to feline T cells chronically infected with a mucosally transmissible FIV strain. WHI-07 gel-microemulsion was a potent vaginal contraceptive in the rabbit model. Both short-term (up to 90 days) and long-term (2-yr) repetitive intravaginal administrations of WHI-07 at concentrations as high as 5.7 ´ 106 times its in vitro anti-HIV IC50 and 5700-times its spermicidal EC50 were not associated with mucosal, systemic, reproductive or developmental toxicity in mice and rabbits and did not produce increased carcinogenicity in mice. WHI-07 has clinical potential as a safe prophylactic contraceptive anti-HIV microbicide for sexually active women. Keywords: contraceptive, microbicide, toxicity, zidovudine

Domestic cat model for predicting human nucleoside analogue pharmacokinetics in blood and seminal plasma.

To establish whether a feline model can predict nucleoside analogue behavior in human semen, zidovudine (ZDV) and lamivudine (3TC) pharmacokinetic parameters (PKs) were determined in the blood and seminal plasma of healthy cats. Our results show considerable similarity in ZDV and 3TC PKs between cats and humans. As in humans, ZDV and 3TC tend to accumulate in feline seminal plasma. Area under the blood plasma concentration-time curve was predictive of seminal plasma excretion. The felid model offers a unique in vivo experimental alternative for investigating the pharmacokinetics of nucleoside analogues in the male genital tract.

Domestic Cat Model for Predicting Human Nucleoside Analogue Pharmacokinetics in Blood and Seminal Plasma

Domestic Cat Model for Predicting Human Nucleoside Analogue Pharmacokinetics in Blood and Seminal Plasma

Zona pellucida piercing enhances zona penetration by spermatozoa from normospermic and teratospermic domestic cats.

Poor quality ejaculates appear associated with reduced zona penetration and poor fertilization in vitro in some felid species or populations. Even morphologically normal sperm from these teratospermic ejaculates are compromised in ability to penetrate the zona pellucida and fertilize oocytes. A domestic cat model and zona piercing were used to study the function of normal and malformed sperm. Male cats naturally producing different proportions of morphologically normal sperm (group I, > 60%; group II, 45-55%; group III, < 40%) were identified. Electroejaculates were subjected to sperm washing (WS) and half of these to swim-up (SU) processing following WS, and all samples were assessed for a sperm motility index (SMI) over time. Zonae of salt-stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Control (nonmanipulated) SS oocytes (n = 430) and ZnPd-SS oocytes (n = 430) were coincubated with sperm in vitro. The proportion of morphologically normal sperm/ejaculate differed (P < 0.05) among groups averaging 68.8% for group I, 53.5% for group II, and 34.7% for group III. After SU processing, the percent normal sperm among groups was similar (P > 0.05; range, 70.8-80.2%). Average SMI for all groups was high (> 70) at 0 hour and decreased over time, but the descent of slope was greater (P < 0.05) for group III than groups I and II. The proportion of oocytes penetrated was not influenced (P > 0.05) by sperm-processing treatment. For all groups, inner zona penetration was greater (P < 0.05) in ZnPd-SS than SS oocytes (group I, 52.3% vs. 34.9%; group II, 37.9% vs. 20.3%; group III, 32.0% vs. 17.2%).(ABSTRACT TRUNCATED AT 250 WORDS)