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Sperm maturation depends on exposure to microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the extracellular vesicles from different regions of the epididymis can ensure paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to extracellular vesicles collected from upstream (i.e., caput) segments. Impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that extracellular vesicles from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to extracellular vesicle supplementation). Of note, expressions of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were influenced by the presence of extracellular vesicles. Together, our findings comprise the first report in any species of paracrine control of segmental gene regulation by epididymal extracellular vesicles. These results contribute to a better understanding of epididymis biology and could lead to strategies to enhance or suppress male fertility.Extracellular vesicles (EVs) from the caput epididymis influenced gene expression, with 1233 differentially expressed genes identified, in corpus epididymis explants after 3 days of exposure to EVs using a reproducible in vitro culture system.Graphical Abstract

Germinal vesicles are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved germinal vesicles can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a germinal vesicle preservation method that employs non-ionizing microwave radiations imparting energy to water molecules, which results in rapid and homogeneous drying of the sample. Trehalose is added as a xero-protectant before the radiations, enabling isothermal vitrification of the disaccharide sugar during drying. While the technique is still considered experimental, studies have shown that DNA and structural integrity can be effectively maintained in dried/rehydrated germinal vesicles. Importantly, the dry-preservation approach allows supra-zero temperature storage of the samples, offering a cost-effective and energy-saving alternative to traditional methods relying on ultra-low freezing temperatures. The protocol outlines a comprehensive procedure involving germinal vesicle oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates the ease of manipulation, making it an accessible method for researchers. While initially developed for domestic cats, the protocol can be adapted for other species with necessary modifications, considering potential species-specific responses to dehydration stress.This is a detailed protocol for microwave-assisted dehydration of GVs to allow fertility preservation and long-term storage at supra-zero temperatures.Graphical Abstract

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.The GM1-enriched vesicles that transfer GM1to the sperm plasma membrane during epididymal transit are highly abundant in segment 2 of the murine epididymis, and their differential presence is regulated by luminal fluid factors.Graphical Abstract

The objective of the study was to evaluate the integrity of cat testicular tissues after vitrification with different devices followed by different warming conditions. The influence of vitroculture for 24 hours after warming also was examined. Testicular tissues from adult domestic cats were dissected in small fragments that were vitrified using Cryotop®or threaded on fine needles, warmed (directly at 37°C or with a preliminary 10 seconds exposure to 50°C), and/or cultured in vitrofor an additional 24 hours. For each treatment group, tissues were assessed based on histology, apoptosis, and sperm DNA integrity. Results showed that fragments of testicular tissues were efficiently cryopreserved (maintaining the quality of all cell types) with vitrification with Cryotop followed by direct warming at 37°C, and additional culture of 24 hours at 38.5°C. These encouraging results are paving the road to optimize preservation protocols and use them for systematic banking of tissues from genetically valuable felids.

There is a remarkable diversity in the animal kingdom regarding mechanisms underlying the production, maturation, structure, and function of sperm cells. Spermatology studies contribute to the knowledge of species diversity and also provide information about individual or population fitness. Furthermore, this fundamental research is required before collected spermatozoa can be used for conservation breeding, including assisted reproduction and cryobanking. This article aims to (a) review the most recent knowledge on sperm morphology and function in wild animal species, (b) analyze how this knowledge can be used to save species in their natural habitat or ex situ, and (c) propose future scientific directions in wildlife spermatology that could positively impact animal conservation. Variations in sperm structure and performance within and between species have multiple origins and significance. This collective body of knowledge enables the design and implementation of conservation strategies and action plans that integrate several disciplines.

Reproductive science in the context of conservation biology is often understood solely in terms of breeding threatened species. Although technologies developed primarily for agriculture or biomedicine have a potentially important role in species conservation, their effectiveness is limited if we regard the main objective of animal conservation as helping to support populations rather than to breed a small number of individuals. The global threats facing wild species include the consequences of climate change, population growth, urbanization, atmospheric and water pollution, and the release of chemicals into the environment, to cite but a few. Reproductive sciences provide important and often unexpected windows into many of these consequences, and our aim here is both to demonstrate the breadth of reproductive science and the importance of basic knowledge and to suggest where some of the insights might be useful in mitigating the problems.

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and/or introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above −10 °C, which enables cryopreservation of hiPSCs with no serum, much-reduced cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by their pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.

Endometrial receptivity and embryo implantation processes are a major point of pregnancy failure in many mammalian species, including humans. Although reproductive biology in many carnivore species remains enigmatic, the few that have been studied so far are invaluable comparative models. The goals of this review are to (1) summarize current data on the mechanisms involved in uterine receptivity and embryo implantation in carnivores, including commonalities and differences with other mammalian species and (2) identify research priorities to better understand a key phenomenon in a critical group of mammals. Besides unique reproductive traits in some carnivores (induced vs. spontaneous ovulation in cats, ovulation at the germinal vesicle stage in dogs), preimplantation embryo development is comparable with other orders. However, the timing of implantation varies, especially in species having an embryonic diapause. Mechanisms involved in endometrial receptivity and decidualization still remain to be fully understood, but specific markers have already been identified. Importantly, the use of endogenous hormones to control the ovarian activity may impact endometrial receptivity and subsequent embryo implantation. Next, research efforts should take advantage of advanced technologies to further study embryo implantation in carnivores and to provide more relevant models to reproductive medicine or for the conservation of rare and endangered species.The goals of this review are to (1) summarize current data on the mechanisms involved in uterine receptivity and embryo implantation in carnivores and (2) identify research priorities to better understand a key phenomenon in a critical group of mammals.

Extracellular vesicles (EVs) isolated from epididymal segments of both normospermic and teratospermic domestic cats were analyzed via mass spectrometry. Both male types shared 3008 proteins, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types, with seven cauda-derived proteins trending downward in teratospermic compared to normospermic males. Collectively, these differences may relate to the poor sperm quality observed in teratospermic individuals.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above −20°C, which is one step closer to future storage at supra-zero temperatures.

To determine the impact of neurotensin (NTS), a naturally occurring peptide, on the function of human and nonhuman primate sperm.

Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial-blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

Because of higher extinction rates due to human and natural factors, more basic and applied research in reproductive biology is required to preserve wild species and design proper strategies leading to sustainable populations. The objective of the review is to highlight recent, inspiring breakthroughs in wildlife reproduction science that will set directions for future research and lead to more successes in conservation biology. Despite new tools and approaches allowing a better and faster understanding of key mechanisms, we still know little about reproduction in endangered species. Recently, the most striking advances have been obtained in nonmammalian species (fish, birds, amphibians, or corals) with the development of alternative solutions to preserve fertility or new information about parental nutritional influence on embryo development. A novel way has also been explored to consider the impact of environmental changes on reproduction-the allostatic load-in a vast array of species (from primates to fish). On the horizon, genomic tools are expected to considerably change the way we study wildlife reproduction and develop a concept of "precision conservation breeding." When basic studies in organismal physiology are conducted in parallel, new approaches using stem cells to create artificial gametes and gonads, innovations in germplasm storage, and more research on reproductive microbiomes will help to make a difference. Lastly, multiple challenges (for instance, poor integration of new tools in conservation programs, limited access to study animals, or few publication options) will have to be addressed if we want reproductive biology to positively impact conservation of biodiversity.

Although the cheetah (Acinonyx jubatus) routinely lives for more than 12 yr in ex situ collections, females older than 8 yr reproduce infrequently. We tested the hypothesis that reproduction is compromised in older female cheetahs due to a combination of disrupted gonadal, oocyte, and uterine function/integrity. Specifically, we assessed 1) ovarian response to gonadotropins; 2) oocyte meiotic, fertilization, and developmental competence; and 3) uterine morphology in three age classes of cheetahs (young, 2–5 yr, n = 17; prime, 6–8 yr, n = 8; older, 9–15 yr, n = 9). Ovarian activity was stimulated with a combination of equine chorionic gonadotropin and human chorionic gonadotropin (hCG), and fecal samples were collected for 45 days before gonadotropin treatment and for 30 days after oocyte recovery by laparoscopy. Twenty-six to thirty hours post-hCG, uterine morphology was examined by ultrasound, ovarian follicular size determined by laparoscopy, and aspirated oocytes assessed for nuclear status or inseminated in vitro. Although no influence of age on fecal hormone concentrations or gross uterine morphology was found (P> 0.05), older females produced fewer (P< 0.05) total antral follicles and oocytes compared to younger counterparts. Regardless of donor age, oocytes had equivalent (P> 0.05) nuclear status and ability to reach metaphase II and fertilize in vitro. A histological assessment of voucher specimens revealed an age-related influence on uterine tissue integrity, with more than 87% and more than 56% of older females experiencing endometrial hyperplasia and severe pathologies, respectively. Our collective findings reveal that lower reproductive success in older cheetahs appears to be minimally influenced by ovarian and gamete aging and subsequent dysfunction. Rather, ovaries from older females are responsive to gonadotropins, produce normative estradiol/progestogen concentrations, and develop follicles containing oocytes with the capacity to mature and be fertilized. A more likely cause of reduced fertility may be the high prevalence of uterine endometrial hyperplasia and related pathologies. The discovery that a significant proportion of oocytes from older females have developmental capacity in vitro suggests that in vitro fertilization and embryo transfer may be useful for “rescuing” the genome of older, nonreproductive cheetahs.

The diet is considered to be the main source of body burden of polychlorobiphenyls (PCBs) in the general population. The most cost-effective and reliable way to estimate the dietary intake is based on total diet studies (TDSs). The aim of the present study was to provide a reliable estimate of recent average dietary intakes of PCBs in Northern Italy. Congener-specific analysis of PCBs was undertaken on total diet samples collected at the cafeteria of the University of Pavia. The average daily intake for total PCBs was calculated to be 0.26 µg/person day-1. The major contributor to PCB intake was accounted for by pooled samples of bread, cereals and potatoes, followed by the protein composite, and fruit and vegetables. Although diverse dietary habits and differences in sampling and analytical procedures make comparison between different studies difficult, the daily intake of PCBs in Northern Italy can be located in the lower part of the range for values reported in the literature.

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.

Sustaining viable populations of all wildlife species requires the maintenance of habitat, as well as an understanding of the behaviour and physiology of individual species. Despite substantial efforts, there are thousands of species threatened by extinction, often because of complex factors related to politics, social and environmental conditions and economic needs. When species become critically endangered, ex situ recovery programmes that include reproductive scientists are the usual first line of defence. Despite the potential of reproductive technologies for rapidly increasing numbers in such small populations, there are few examples of success. This is not the result of a failure on the part of the technologies per se, but rather is due to a lack of knowledge about the fundamental biology of the species in question, information essential for allowing reproductive technologies to be effective in the production of offspring. In addition, modern conservation concepts correctly emphasise the importance of maintaining heterozygosity to sustain genetic vigour, thereby limiting the practical usefulness of some procedures (such as nuclear transfer). However, because of the goal of maintaining all extant gene diversity and because, inevitably, many species are (or will become) ?critically endangered', it is necessary to explore every avenue for a potential contributory role. There are many ?emerging technologies' emanating from the study of livestock and laboratory animals. We predict that a subset of these may have application to the rescue of valuable genes from individual endangered species and eventually to the genetic management of entire populations or species. The present paper reviews the potential candidate techniques and their potential value (and limitations) to the study and conservation of rare wildlife species.

After in vitro fertilization with spermatozoa from bulls with high in vitro fertility, a beneficial paternal effect is manifested during the G1 phase of the first cell cycle. This benefit determines an earlier onset of the first S phase, and then a successful morula-blastocyst transition 7 days later. We hypothesized that the origin of the paternal effect could be a shift of the metabolism of the fertilized oocyte, because in mice, sperm decondensation is responsible for a dramatic increase in glucose metabolism. In this study we investigated the interaction between both pronuclei and compared glycolysis and pentose phosphate pathway (PPP) activities in bovine oocytes fertilized with spermatozoa from bulls of high or low fertility. Here we demonstrate that male pronucleus formation is necessary for the onset of the S phase in the female pronucleus, and that the component promoting an early S phase in both pronuclei is metabolic and linked to an up-regulation of the PPP during the male pronucleus formation. This long-lasting paternal effect is more evidence of the important role of epigenetic control during early embryo development.

Assisted reproductive techniques (gamete cryopreservation, artificial insemination, embryo transfer, and in vitro fertilization) allow to propagate small fragmented populations of wild endangered species or domestic breeds. There are the best way for producing several offspring from selected genitors in order to avoid inbreeding depression. However, few mammalian species have been well studied for their reproductive biology whereas huge differences have been observed between these species. Furthermore, materials, methods and experimental designs have to be adapted for each case and each limiting factor (wildness, poor quantity of biological material, disparate locations). Genome resource banking is currently arising and the most applied reproductive biotechnology remains artificial insemination. Assisted reproductive techniques currently developed in domestic species (intra-cytoplasmic sperm injection, nuclear transfer) may offer new opportunities for the propagation of endangered species.

The aim of this study was to characterize the respective influences of the paternal and the maternal components on the timing of the first S-phase in the bovine zygote. In vitro-matured oocytes were fertilized in vitro with sperm conferring a high blastocyst rate (embryos of group 1) or a low blastocyst rate (embryos of group 2). Resulting zygotes were either allowed to develop in vitro to the blastocyst stage or exposed to 5′-bromo-2′-deoxyuridine in order to characterize the timing of their first S-phases. Timing of pronuclear formation was similar in the two groups, but the onset of S-phase and the first cleavage occurred earlier in group 1 than in group 2. We also showed that the length of the S-phase represented 30% of the first cell cycle in group 1 and 20% in group 2. Differences in times of onset of the first S-phase observed between embryo groups concerned both male and female pronuclei in a similar manner and were not dependent on the maternal component of the zygote. Our data demonstrated that the precocity of the onset of the first S-phase stemmed from a paternal control exerted during a transient period of the G1-phase.

The electrochemical aspects of metal etching to form patterned conductors and of corrosion of conductors in the field are closely related. Both need to be considered in designing metallization structures for microelectronic devices. The evolution of a manufacturing process for a multi-level interconnect structure is discussed from an electrochemical perspective. A galvanic corrosion problem during manufacture and its solution for the interconnect metallization on a silicon integrated circuit are also discussed. Following the discussion on etching processes and corrosion during manufacture, a discussion of electrochemical and electrolytic failure mechanisms for electronic equipment in field environments and some steps that can be taken to prevent harmful environmental effects are presented. Recent research on the adhesion of various protective coatings and interlevel polymeric dielectrics is presented in the context of failure prevention.

Ex situmanagement of the cheetah, an induced ovulator, benefits from assisted reproductive technology that requires equine chorionic gonadotropin (eCG) to stimulate follicular development and human chorionic gonadotropin (hCG) to provoke nuclear maturation/ovulation. We tested two hypotheses: 1) cheetah ovarian and follicular metrics (volume, number, vascularization) post-gonadotropins correlate to oocyte quality, specifically nuclear status and post-fertilization developmental competence; and 2) such metrics are useful for developing a viable mathematical index to predict oocyte quality. Female cheetahs (n = 28; age, 2-15 yr) were given 300 IU eCG (i.m.) followed 82-86 h later by 150 IU hCG (i.m.). Twenty-six to 32 h post-hCG, total follicle number (≥ 2 mm diameter), ovarian volume (ellipsoid shape [(4 x π x 0.5 height x 0.5 length x 0.5 width)/3]), individual follicular volume [(π x follicle diameter)3/6] and follicular vascularization (score 0 - 3; 3 = most vascular) were determined by laparoscopy. The combined ovarian volume (Ov Vol), average follicular volume (Fl Vol) and average vascularization score (Fl Vasc) were calculated for each female. Oocytes were recovered by aspiration and a cohort from each animal immediately fixed for nuclear maturation assessment. Remaining oocytes were inseminated in vitrowith ≈1 x 106frozen-thawed, motile sperm/ml and cultured in Hams F10 with 5% fetal calf serum in 5% CO2 in air. After a 7-day culture, embryos were fixed and stained to count blastomeres. Ovarian and follicular metrics were analyzed using Pearson correlations in relation to oocyte maturation (at aspiration; proportion at metaphase II, MII) and developmental success to 8-cell embryos following fertilization (% 8 cells). The metric Ov Vol correlated positively with total follicle number (R2= 0.463, P< 0.01) and total oocytes recovered (R2= 0.500, P< 0.01), whereas Fl Vol correlated positively with % MII oocytes recovered (R2= 0.444, P< 0.02) and % 8-cell embryos (R2= 0.444, P< 0.05), all regardless of donor age. Additionally, Fl Vasc positively correlated with % MII oocytes (R2= 0.370, P< 0.05) for all females. Based on significant correlations and to investigate the ability of ovarian and follicular metrics to be used as indicators of oocyte quality, the following regression model was fit to the data using step-wise selection criteria: oocyte quality = β0+ β1[(Fl Vol/Ov Vol) x Fl Vasc + follicle number] + β2female age + ϵ, where ϵ represents residual error. For all tested models, factors with P< 0.60 entered the step-wise model, and values with P< 0.10 remained in the model. The following combination of factors [(Fl Vol/Ov Vol) x Fl Vasc + follicle number] significantly predicted the reproductive outcomes of % MII oocytes recovered (R2= 0.2124, P< 0.01, C(p) = 1.6015) and % 8-cell embryos produced (R2= 0.3288, P< 0.01, C(p) = 1.9191). Together, these data indicate the potential of predicting oocyte quality (maturation and developmental competence) by integrating and modeling easily-measurable traits (via laparoscopy) that are associated with ovarian and follicular morphology. Although individual characteristics are insufficient to calculate oocyte quality, combining multiple factors into the comprehensive index [(Fl Vol/Ov Vol) x Fl Vasc + follicle number] allows estimating the likely potential of cheetah oocytes to achieve nuclear maturation and develop to late-stage embryos in vitro.

Cytoplasmic defects as a result of aging or cryopreservation can be circumvented in immature oocytes by transferring the germinal vesicle (GV) into a previously enucleated good quality oocyte. We are interested in oocyte reconstruction by GV transfer in felids to provide options for propagating rare domestic cat biomedical models as well as endangered species. Objectives of this study were to assess the impact of (1) cumulus cell removal, (2) treatments that allow viewing the GV, and (3) enucleation and fusion on subsequent oocyte meiotic resumption. In Experiment 1 (3 replicates), grade I immature cumulus-oocytes complexes (COC) collected from adult cat ovaries were denuded in 0.2% hyaluronidase and matured in vitro (28 h) alone (n = 56 total denuded oocytes; DO) or in co-culture (n = 52 DO with equal number of COC). Nuclear status of DO and intact COC was assessed by fixation in ethanol and Hoechst 33341 staining. The proportions of metaphase II stage (MII) oocytes was less (P< 0.05; ANOVA) when oocytes were denuded and matured alone (23.8 ± 2.7%; Mean ± SD) compared to the co-cultured DO (76.7 ± 4.3%) or intact COC (82.3 ± 3.1%). In Experiment 2 (3 replicates), oocytes were denuded and either treated to visualize the GV (n = 74; incubation in 7.5 μg/ml Cytochalasin B for 15 min at 38.5°C, centrifugation at 15,000 × gfor 10 min, and exposure to 0.1 M sucrose for 10 min at 38.5°C) or not treated (n = 75; control). Nuclear status of treated and control DO was assessed after in vitro maturation (IVM) with intact COC (co-culture; ratio 1:1). Percentages of MII were similar (P> 0.05) between DO treated to visualize the GV (79.7 ± 3.5%) and controls (82.7 ± 4.6%). In Experiment 3 (4 replicates), oocytes were denuded and treated as described above. After partial zona dissection of DO and enucleation, GV surrounded by a small amount of cytoplasm (karyoplasts) were fused by two electrical pulses (1.8 kV/cm DC for 30 μs in 0.15 M sorbitol) with the same or different cytoplasts (n = 93 homoplasmic, 91 heteroplasmic reconstructions, respectively). Control oocytes (n = 85) were denuded, treated to visualize the GV and subjected to electric pulses alone. Nuclear status of reconstructed and control oocytes was assessed after IVM with intact COC (1:1). Percentages of fused oocytes after homo- (71.7 ± 6.3%) and heteroplasmic reconstruction (69.0 ± 4.2%) were similar (P> 0.05). The proportion of fused oocytes reaching MII after homo- (46.5 ± 2.2%) versus heteroplasmic (47.7 ± 1.4%) reconstruction also did not differ (P> 0.05), but was less (P< 0.05) than controls subjected to electric pulses alone (80.0 ± 3.5%). This is the first report in carnivores of successful oocyte reconstruction by GV transfer. Results clearly demonstrate that removing cumulus cells is detrimental to nuclear maturation, but can be circumvented by co-culturing DO with intact COC. Furthermore, treatments that allow viewing the GV have no impact on oocyte nuclear maturation. Although enucleation reduces the overall proportion of oocytes able to reach MII, neither electric pulses nor heteroplasmic reconstruction appear to have any influence on GV-cytoplasm interactions during meiotic resumption. (Supported by the National Institutes of Health K01 RR020045). (platform)

Histone deacetylase 2 (HDAC2) is a key transcriptional coregulator that is suspected to play a role during oogenesis. It is known that RNA transcription in the cat germinal vesicle (GV) stops during folliculogenesis at the late antral follicle stage and is unrelated to histone deacetylation or chromatin condensation. The objective of the present study was to determine if and how HDAC2 participates in transcription regulation in the cat GV. Spatiotemporal HDAC2 protein expression was examined by immunostaining oocytes from primary to large antral follicles. HDAC2 was detected in the majority of GVs within oocytes from early, small, and large antral follicles. At early and small antral stages, HDAC2 was found primarily in the GV's nucleoplasm. There then was a significant shift in HDAC2 localization into the nucleolus, mostly in oocytes from large antral follicles. Assessments revealed that transcription was active in oocytes that contained nucleoplasm-localized HDAC2, whereas nucleolar-bound HDAC2 was associated with loss of both global transcription and ribosomal RNA presence at all antral stages. When oocytes were exposed to the HDAC inhibitor valproic acid, results indicated that HDAC regulated transcriptional activity in the nucleoplasm, but not in the nucleolus. Collective results suggest that nucleolar translocation of HDAC2 is associated with transcriptional silencing in the GV, thereby likely contributing to an oocyte's acquisition of competence.

This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (age, <6 mo) versus adult (age, >8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P< 0.05) follicle viability in prepubertal donors in a dose-dependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by coincubation with an inhibitor of EGF receptor (AG1478), mitogen-activated protein kinase (MAPK; U0126), or phosphoinositide 3-kinase (PI3K; LY294002). EGF enhanced (P< 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P< 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying influence of age in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females.

The study explored a novel approach for preserving the maternal genome without the entire oocyte by air-drying the cat germinal vesicle (GV) in the presence of the disaccharide trehalose. Specifically, we examined GV structure and function after desiccation, storage at 4°C (up to 32 wk), and rehydration including the ability to resume meiosis after injection into a fresh, conspecific cytoplast. In experiment 1, DNA integrity was similar to fresh controls after 1 and 4 wk storage in the presence of trehalose, but was more fragmented at later time points (especially after 32 wk). Nuclear envelope integrity was sustained in >90% of oocytes stored for 0, 4, or 16 wk regardless of protective treatment. In experiment 2, compacted, air-dried GVs were stored for 2 or 4 wk, rehydrated, and injected into fresh cytoplasts. After culture for 24 h in vitro, up to 73% of oocytes reconstructed with desiccated GVs preserved in trehalose resumed meiosis compared to 30% of those dried in the absence of the disaccharide. At each storage time point, trehalose presence during air-drying was advantageous for resumption of meiosis, with >20% of oocytes completing nuclear maturation to metaphase II. This demonstrates a potential for preserving the female genome using the GV alone and for multiple weeks after desiccation. Trehalose enhanced the process by retaining the ability of a dried and rehydrated GV to resume communication with the surrounding cytoplasm of the recipient oocyte to permit reaching metaphase II and likely sustain subsequent embryo development.

The estrous cycle of the cheetah, an induced ovulator, occurs about every 14 days. Therefore, the ovary of this species almost is constantly experiencing folliculogenesis and producing surges or waves of estrogen activity. Elevated estrogen compromises the effectiveness of exogenous gonadotropins used to time assisted breeding. Approximately 35% of female cheetahs fail to respond to exogenous gonadotropins, and oocyte quality is compromised when such stimulation commences during an endogenous follicular phase. Given the general sensitivity of felids to progesterone and the comparatively short reproductive cycle of the cheetah, we hypothesized that even a brief exposure (7 days) to a low-dose oral progestin (Altrenogest) would suppress ovarian activity. Cheetah females (age range, 5-11 y) were given either 0.1 mg/kg (n=3, low dose) or 0.4 mg/kg (n=3, high dose) Altrenogest for 7 days. Fecal samples were collected (4-5/wk/female) for 41 to 44 days before, 7 days during and 33 to 36 days after Altrenogest treatment. Baseline fecal estradiol concentrations were calculated for each female using an iterative process, excluding values that exceeded the mean + 1.5 SD. An estradiol peak was the highest value within a group of samples > 1.5 x baseline. An estrous cycle was defined as the interval between two consecutive estradiol peaks = 4 days apart. Data were analyzed by GLM procedures and expressed as LSmeans ± SEM. Females receiving the low Altrenogest dose experienced a longer (P< 0.05) duration from the end of progestin exposure to the next post-treatment estradiol peak (13.3 ± 2.8 days) compared to those receiving the high dose (2.0 ± 2.8 days). For the 5 weeks after treatment, cheetahs receiving the higher Altrenogest dose also had an increased (P< 0.05) frequency of estrous cycles as measured by number of estradiol peaks (5.7 ± 0.7 cycles) compared to those receiving the lower dose (2.3 ± 0.7 cycles over the same time period). Thus, a short administration of oral progestin quickly down-regulated ovarian activity, probably because the induced-ovulating cheetah with naturally short estrous cycles rarely is exposed to progestagens and, therefore, is highly sensitive. The higher Altrenogest dose appeared to stimulate folliculogenesis, perhaps related to a strong endogenous FSH rebound post-progestin withdrawal. These findings have practical application as a simple, short-term approach for suppressing ovarian activity in anticipation of generating a more consistent gonadotropin induction response for timed artificial insemination or embryo transfer.

Immature cat oocytes collected during seasonal anestrus (August-January) require high FSH concentrations in vitroto achieve nuclear and cytoplasmic maturation. The normally compromised meiotic and developmental competence of these oocytes is not due to reduced FSH-receptor (FSHR) abundance. To better understand the physiological mechanisms of this seasonal difference, we assessed the impact of the seasonal anestrus on oocyte nuclear and cytoplasmic maturation in vivoas well as the expression of maturation-related genes. Twelve adult cats were exposed for 3 to 5 months to a natural breeding season (14 hours of light and 10 hours of dark) and then to a similar interval simulating anestrus (8 hours of light and 16 hours of dark). After ovarian stimulation with exogenous eCG/hCG, oocytes were aspirated from preovulatory follicles by laparoscopy. Grade 1 cumulus-oocyte complexes (COCs; dark oocyte cytoplasm and several layers of expanded cumulus cells) were selected and inseminated (n = 30 during the breeding season; n = 45 during anestrus) with frozen-thawed sperm. Presumptive embryos then were cultured for 8 days, fixed in 70% v/v ethanol and stained with Hoechst to determine the number of blastomeric nuclei. During each interval, a proportion of Grade 1 COCs (n = 8 during the breeding season; n = 11 during anestrus) were collected for relative quantification of maturation-related genes (ESR2, PTGS2, EGFR). Total RNA was extracted using a Picopure RNA isolation kit and cDNA generated using random primers and Superscript III reverse transcriptase. cDNA samples were assessed by real-time qPCR to compare relative expression levels of FSHR, ESR2, PTGS2and EGFR(normalized to ACTB, RPS7and YWHAZexpression). Proportions of Grade 1 COCs at the metaphase II stage were similar between the two intervals. Relative expression levels of developmental markers ESR2, PTGS2, EGFRalso were comparable. A trend for decreased EGFRexpression was associated with a lower proportion of oocytes fertilized and reaching an advanced embryo stage (P<0.05) during anestrus compared to the breeding season. Therefore, although oocyte meiotic competence is unimpaired in vivo, oocyte developmental competence is compromised during the anestrual compared to the breeding season. Generally, this effect on developmental capacity was not reflected in all maturation-related genes. Collective results infer that alternate functions of FSH unrelated to downstream gene regulation (such as EGFR) may be responsible for seasonal decreases in oocyte developmental capacity in vivo. Further investigation of FSHR functionality will identify alternate mechanisms to manipulate FSH responsiveness in vivo. (Supported by the Kelly Fellowship and Lalor Foundation)(poster)

The ability to compact and inject the cat germinal vesicle into a recipient cytoplast allows exploration of a new fertility preservation strategy that could avoid the need to conserve whole oocytes. Similar to sperm desiccation, the drying and storage of germinal vesicles at 4°C could be an alternative to classical oocyte cryopreservation methods. The objective was to analyze the protective effect of trehalose on the structure and function of the desiccated germinal vesicle of the domestic cat. Specifically, this study investigated (1) the integrity of germinal vesicle DNA and the presence of the nuclear envelope after desiccation and rehydration and (2) the germinal vesicle's ability to resume meiosis after desiccation, rehydration and injection into a fresh, conspecific cytoplast. In Experiment 1, immature oocytes were denuded of cumulus cells, permeabilized with hemolysin and then exposed to 0 or 1.5 M trehalose before air-drying on a glass slide. After 0, 1, 2, 4 or 8 weeks of storage at 4°C, oocytes (n = 1,743 in 5 replicates) were rehydrated and fixed to assess the incidence of DNA fragmentation (Comet assay and TUNEL assay), and the presence of the nuclear envelope (lamin A/C immunodetection). At each time point, oocytes desiccated with or without trehalose were compared to fresh controls. The beneficial effect of trehalose on DNA integrity was significant (there were no differences with controls) at 1 and 4 weeks of storage; however, DNA was more fragmented at all other time points regardless of the presence or absence of trehalose. This finding was unrelated to nuclear envelope damage that was only observed in oocytes stored for 0 or 4 weeks. Based on these results, Experiment 2 was designed to explore the functionality of the germinal vesicle at 2 weeks (where there was no apparent benefit of trehalose) and 4 weeks (where there was a benefit of trehalose) of storage. Immature cat oocytes (n = 180 in 3 replicates) were denuded, and exposed to 1 mM resveratrol to compact the germinal vesicle. Oocytes then were permeabilized and exposed to 0 or 1.5 M trehalose before air-drying. At 2 or 4 weeks of storage, germinal vesicles were rehydrated and injected into fresh cytoplasts. Reconstructed oocytes (including a control group injected with fresh germinal vesicles) were cultured in vitro for 24 hours in standard maturation conditions before fixation and staining to assess germinal vesicle survival (chromatin re-expansion) and meiotic resumption. Desiccation reduced germinal vesicle survival and meiotic resumption compared to fresh controls; however, within each time point, trehalose presence during desiccation significantly increased both germinal vesicle survival as well as the ability to reach the metaphase II stage. There was no difference in germinal vesicle functionality observed between 2 and 4 weeks of storage. Under optimal treatment conditions, as many as 60% germinal vesicles survived and 10% reconstructed oocytes achieved nuclear maturation. In conclusion, critical germinal vesicle components are preserved for at least 4 weeks in the presence of trehalose. This disaccharide also preserves the ability of a desiccated germinal vesicle to resume meiosis once rehydrated and injected into a fresh cytoplast. This research was supported by the Smithsonian Scholarly Studies Program and the National Institutes of Health (R01 RR026064).(poster)

Sustaining viable populations of any wildlife species requires a combination of adequate habitat protection as well as a good understanding of environmental and biological factors (including reproductive mechanisms) that ensure species survival. Thousands of species are under threat of extinction due to habitat loss/degradation, over-exploitation, pollution, disease, alien species invasions and urban sprawl. This has served as incentive for intensive management of animal populations, both ex situ (in captivity) and in situ (living in their natural habitat). Assisted reproductive technologies developed for addressing human infertility and enhancing livestock production have shown encouraging promise for wildlife species. However, species-specific physiological variations and a lack of fundamental knowledge have limited how these tools can be used to help rapidly re-build sustainable populations of endangered species. Despite limitations, there is enormous potential in applying human-related fertility preservation strategies to wild animals, especially approaches that could assist managing or "rescuing" the genomes of genetically valuable individuals. Indeed, one of the highest priorities in wildlife ex situ management is sustaining all existing genetic diversity to (1) preserve heterozygosity to avoid inbreeding depression and (2) ensure species integrity and the persistence of genomic adaptability to environmental changes. There are specific components of the rapidly emerging field of fertility preservation in men and women that are highly compatible with preserving valuable genomes of individuals or populations of threatened wildlife. Besides the more "classical" approaches focusing on sperm and oocyte freezing, strategies associated with gonadal tissue cryopreservation and in vitro culture are especially attractive for better protecting and extending fertility for wild individuals. Likewise, lessons learned over the last decades in wildlife reproductive biology (either from wild or captive populations) are highly relevant to the advancement of human health and fertility. The substantial amount of scholarly knowledge generated by multispecies and comparative approaches is critical to better understand and mitigate complex issues affecting human beings (fertility, contraception, impact of the environmental changes). Additionally, studies conducted at the molecular or cellular level always are related to physiological investigation in wild individuals or entire populations and take into account the interactions with the environment. Comparative approaches in fertility preservation could benefit to the intensive and practical management of gene diversity in endangered species and lead to translational tools for human reproductive medicine. Our long term support from the National Institutes of Health as well as our current collaborations with specialists in human reproductive medicine perfectly illustrates this concept.(platform)

Cytoplasmic defects in immature oocytes can be circumvented by transferring the germinal vesicle (GV) into a previously enucleated, good quality oocyte. However, the karyoplast containing the large donor GV must be fused with the recipient's cytoplast membrane, which results in a reconstructed oocyte with problematic heteroplasmy and impaired capacity to achieve nuclear maturation. Using the ability to artificially compact the GV in the cat model, the objective was to assess the impact of GV transfer by fusion of a smaller donor GV-karyoplast or by intracytoplasmic GV injection on (1) maintenance of homoplasmy and (2) nuclear maturation in the reconstructed oocyte. In Experiment 1 (3 replicates), immature cat oocytes were prepared for micromanipulation (cumulus removal, centrifugation and Cytochalasin B exposure). In each replicate, donor oocyte mitochondria were labeled (400 nM fluorescent Mitotracker Green; 15 min) before transferring the GV into a recipient cytoplast by: 1) membrane fusion (induced by Sendai virus); 2) membrane fusion after GV compaction with 1 mM resveratrol (1 h); or 3) intra-cytoplasmic injection (using a 15 μm diameter Piezo-actuated pipette) after GV compaction, aspiration in the pipette and extensive washing (n = 12 transfers/treatment). Diameters of donor GVs and resulting GV-karyoplasts were measured during each transfer. After membrane fusion or injection, reconstructed oocytes were fixed to assess the presence of labeled mitochondria. Diameters of donor GVs or GV-karyoplasts were smaller (P<0.05; ANOVA) after compaction (17.1 ± 2.0 μm and 21.6 ± 2.4 μm, respectively; mean ± SD) compared to without (41.5 ± 3.0 μm and 48.4 ± 5.4 μm, respectively). No fluorescence was detected after GV injection, whereas oocytes reconstructed by fusion always contained labeled mitochondria indicative of heteroplasmy regardless of volume of the donor GV-karyoplast. In Experiment 2 (4 replicates), oocytes were prepared for micromanipulation as above. Each replicate included GV transfers by fusion, fusion after compaction or injection after compaction (n = 20 transfers/method) as well as control oocytes that were either left intact, GV compacted, sham injected or GV compacted plus sham injected (n = 20/control group). After in vitro maturation (28 h), all oocytes were fixed to evaluate the efficiency of the GV transfer method as well as nuclear stage and metaphase II spindle integrity. GV injection resulted in more successful reconstructions (95.7 ± 3.1%; P<0.05) compared to fusions with large (74.4 ± 5.3%) or smaller (73.2 ± 3.3%) donor GV-karyoplasts. Proportions of reconstructed oocytes at the metaphase II stage were higher (P<0.05) after GV injection (71.7 ± 3.4%) than after fusion with large (59.6 ± 4.5%) or smaller (57.1 ± 6.0%) donor GV-karyoplasts, but were lower (P<0.05) than in all controls (range, 77.8 to 82.0%). Regardless of treatment (including controls), percentages of normal metaphase II spindles were similar (range, 83 to 87%; P>0.05). Results demonstrated that the cat GV retains meiotic competence after compaction and injection into a recipient cytoplast. Compared to membrane fusion, even with a reduced-volume GV-karyoplast, intracytoplasmic GV injection prevents heteroplasmy and allows a higher incidence of oocyte reconstruction and nuclear maturation. This approach offers new opportunities for addressing infertility and preserving the maternal genome.(platform)

Successful in vitromaturation (IVM) of cat oocytes can only be achieved during the non-breeding season (September through January) with a 10 times higher concentration of follicle-stimulating hormone (FSH; 10 μg/ml) compared to the breeding season (February through August). Despite the reduced sensitivity to FSH during this anestrual interval, we have observed an increase in expression of full-length FSH receptor (FL-FSHR) mRNA, but not the truncated version (Tr-FSHR), in immature cumulus-oocyte complexes (COCs). The objective of this study was to assess the impact of season on overall FSHR, FSHRsplice variants (FL-FSHR, Tr-FSHR) and estrogen receptor 2 (ESR2; as estrogen is known to synergize with FSH during oocyte maturation) mRNA following IVM (using 1 μg/ml versus 10 μg/ml FSH). Immature Grade 1 COCs (dark oocyte cytoplasm and compacted cumulus cells) were collected from adult cat ovaries in the breeding (Br) and non-breeding (NBr) seasons. The COCs were stored immediately in RNAlater (Br, n = 45; NBr, n= 47; in 3 replicates per season) or after IVM with 1 µ g/ml (IVM1; Br, n = 59; NBr, n = 50) or 10 µg/ml (IVM10; Br, n = 54; NBr, n = 54) FSH for 26 hr. Total RNA was extracted using a Picopure RNA isolation kit and cDNA generated using random primers and Superscript III reverse transcriptase. cDNA samples were assessed by real-time qPCR to compare relative expression levels of overall FSHR, FL-FSHR, Tr-FSHRand ESR2mRNAs (normalized to ACTB, RPS7and YWHAZmRNA expression). The significance of expression ratios was tested using the Pair-Wise Fixed Reallocation Randomization Test. Compared to immature COCs, overall FSHRmRNA expression was decreased (P< 0.01) after IVM1 and IVM10 in the breeding season; but remained similar (P> 0.05) after IVM10 during the non-breeding season. FL-FSHRmRNA expression was decreased (P< 0.01) after IVM1 and IVM10 compared to immature COCs in both seasons. Tr-FSHRmRNA expression was decreased (P< 0.05) after IVM1 and IVM10 compared to immature COCs in the non-breeding season, but was similar (P> 0.05) after IVM10 during the breeding season. ESR2mRNA was decreased (P< 0.01) after IVM in all treatment groups except after IVM10 during the non-breeding season (P> 0.05). Results indicate that (1) FSH-regulated expression of overall FSHR, FL-FSHRand Tr-FSHRmRNAs differ seasonally, (2) increased overall FSHRassociated with a decrease in Tr-FSHRmRNA expression may be necessary for optimal maturation of oocytes during the non-breeding season and (3) deficits in COC sensitivity to FSH during seasonal anestrus may be overcome by increased ESR2mRNA expression. Findings suggest that differential regulation of FSHRmRNA splice variants (FL-FSHR, Tr-FSHR) and ESR2mRNA appears important in FSH-induced oocyte maturation in vitroduring the non-breeding season. (Supported by the Kelly Fellowship and Lalor Foundation).(poster)

Reproductive success is low in cheetahs managed in captivity, especially in females ≥ 9 y of age. This study examined age-related reproductive fitness, testing the hypothesis that aged cheetahs experience reduced ovarian activity and poorer oocyte quality than younger counterparts. The objectives were to evaluate: 1) estrous cyclicity before eCG stimulation (assessed by fecal estrogens); 2) formation of functional corpora lutea (CL) after oocyte aspiration (measured by fecal progestagens, PG); and 3) correlation of PG to oocyte quality. Cheetahs were divided into three age classes (young, 2-5 y, n = 8; adult, 6-8 y, n = 6; aged, 9-15 y, n = 5) and given 300 IU eCG (i.m.) followed 82 to 86 h later by 150 IU hCG (i.m.). Fecal samples were collected (4-5/week/female) for 45 to 47 days before and 32 to 35 days after eCG/hCG. At 26 to 32 h post-hCG, total follicle number (≥ 2 mm diameter) and vascularization (score 0-3; 3 = most vascular) were determined by laparoscopy, and oocytes were recovered by aspiration. Oocyte quality was assessed by grade (1-4, 1 = excellent) and fertilizing ability. Oocytes were inseminated with cryopreserved sperm and cultured in Ham's F10 with 5% fetal calf serum. Fecal samples were lyophilized and boiled in ethanol to extract steroid hormones for EIA analysis. Baseline hormonal concentrations were calculated using an iterative process for each female, excluding values that exceeded the mean + 1.5 SD. An estrogen peak was the highest value within a group of samples > 1.5 x baseline. Estrous cycles were defined as the interval between estrogen peaks ≥ 5 days apart. Anestrous periods were defined as those with no estrogen peaks for > 28 days. Data were analyzed by Pearson correlations and GLM procedures. There was no effect (P> 0.05) of age on the average number of estrogen peaks before eCG (range, 3-4 peaks) or the average number of days between peaks (range, 13-17 d). Females in each age group exhibited anestrous periods, with no differences (P> 0.05) among groups. Young cheetahs produced more (P< 0.05) follicles in response to eCG/hCG than aged females, but follicle number was not correlated (P> 0.05) with estrogen concentrations before eCG. Post-aspiration luteal PG concentration did not correlate with follicle number, but was positively related to vascularization score (r = 0.57, P< 0.05) and number of grade 1 and 2 oocytes recovered (r = 0.59, P< 0.05). There also was a positive relationship between PG concentration after oocyte recovery and percentage of fertilized oocytes that cleaved in vitro (r = 0.82, P< 0.05), although this proportion did not differ (P> 0.05) among age groups. There was no influence (P> 0.05) of age on follicular vascularization, oocyte quality grade or PG concentration after aspiration. Results demonstrate that the fertility decline in older cheetahs is not due to a compromised ability to produce normal ovarian steroid concentrations or patterns. Younger cheetahs are capable of producing more follicles (and, thus, more oocytes) in response to eCG/hCG, but oocyte quality is comparable among age groups. This similarity in ovarian steroid and gamete capacity indicates that cheetah age-related infertility has another etiology, perhaps related to as yet undetermined physiological declines. The recovery of high quality, fertilizable oocytes from older cheetahs offers opportunities for rescuing genetic material from unrepresented individuals.(poster)

Reproductive seasonality in the cat is mediated by photoperiod, with longer daylength associated with increased ovarian activity. In the northern hemisphere, estrus occurs from February through August (breeding season) followed by a seasonal anestrus from September through January (non-breeding season). We have previously observed reduced in vitromaturation and in vitrofertilization success in domestic cat oocytes recovered during the non-breeding season. Subsequently, we demonstrated that Grade 1 cumulus-oocyte complexes (COC; a homogenous dark oocyte surrounded by several layers of compacted cumulus cells) collected during this interval of seasonal quiescence require a 10 times higher follicle-stimulating hormone (FSH) concentration to achieve normal meiotic and cytoplasmic maturation in vitro. The objective of the current study was to better understand this seasonal decreased sensitivity to FSH by comparing the expression of FSH receptor (FSHR) mRNA between the breeding and non-breeding season in (1) Grade 1 COCs and (2) whole ovaries (reflecting mRNA expression of the entire intraovarian follicular population). Ovariectomies were conducted on adult cats in June and July (breeding season; Br) and November through January (non-breeding season; NBr). Within each season, ovaries (n = 6 in Br, n = 10 in NBr) were sliced to isolate Grade 1 COCs (n = 12 in Br, n = 11 in NBr) that were pooled before total RNA extraction using a Picopure RNA isolation kit. For whole ovary assessment, six ovaries (n = 3 in Br, n = 3 in NBr) from six females (1 ovary/female) were snap-frozen for total RNA extraction using Trizol reagent. cDNA was generated from COCs and whole ovarian total RNA using random primers and Superscript III reverse transcriptase. Real-time quantitative polymerase chain reaction (qPCR) was performed to compare relative expression levels of FSHRmRNA from pooled cDNA samples from the breeding and non-breeding seasons. All data were normalized to ACTBmRNA expression levels. The significance of expression ratios of FSHRmRNA was tested using the Pair Wise Fixed Reallocation Randomization Test. Results indicated a 2.8-fold increase (P< 0.01) in FSHRmRNA in Grade 1 COCs and a 1.8-fold increase (P< 0.01) in FSHRmRNA expression in whole ovaries during the non-breeding compared to the breeding season. These results suggest that increased FSHRmRNA expression in Grade 1 COCs during the non-breeding season is related to the decreased sensitivity of Grade 1 COCs to FSH previously described during the non-breeding season. A similar trend in expression in whole ovaries indicates FSHRmRNA levels also may be seasonally regulated in the entire intraovarian follicular population. Our results demonstrate for the first time that the seasonal decrease in sensitivity to FSH in intraovarian cat oocytes is not related to decreased FSHRmRNA expression. We propose that this seasonal increase in FSHRmRNA is either due to (1) an increase in expression of a truncated form of FSHRmRNA which may be translated into a non-functional FSHR or (2) an accumulation of FSHRmRNA in the cytoplasm without translation into FSHR protein. Experiments are in progress to analyze mRNA isoform and protein expression as well as FSHR functionality in the breeding versus non-breeding season.(poster)

Due to certain similarities in reproductive mechanisms, the domestic cat has become a valuable model for exploring fertility preservation strategies for young girls and women. The objective of this study was to assess the survival of primordial follicles in prepubertal and adult ovarian tissues during slow freezing or vitrification. Freshly-excised ovaries from prepubertal (4-6 mo; n = 12 total ovaries; 6 replicates) and adult (>2 yr; n = 12 total ovaries; 6 replicates) females were transported at 4°C before processing. In each replicate, the cortex of each ovary (n = 2 from prepubertal and n = 2 from adult cats) was dissected and cut into 18 pieces (1-2 mm3) that were allocated to different treatments. Three pieces of each cortex were directly fixed (4% paraformaldehyde) to determine primordial follicle density in histological sections (i.e., number of follicles with visible oocyte nuclei/mm2of section) as well as the proportion of intact primordial follicles (i.e., number of oocytes and surrounding granulosa cells without pycnotic nuclei, shrunken cytoplasm or vacuoles relative to a total of 60 follicles examined per ovary). Three other cortical pieces were dissociated (1 mg/ml collagenase) and stained (2 μM calcein-AM and 5 μM ethidium) to evaluate the proportion of viable primordial follicles (i.e., number of oocytes with surrounding granulosa cells stained only with calcein-AM relative to a total of 60 follicles examined per ovary). Six cortical pieces were exposed to 1.5 M ethylene glycol (EG; 15 min) and frozen using a slow cooling device (−1°C/min to −80°C ) before plunging into liquid nitrogen. The remaining six pieces were exposed to 1.5 M EG + 1.5 M DMSO + 0.5 M sucrose (5 min) before vitrification in liquid nitrogen. After 1 week of storage, all samples were rapidly warmed to 38°C and diluted stepwise in decreasing sucrose concentrations before fixation for histological assessment or dissociation for viability staining (as above). Primordial follicle density was higher (P<0.05; ANOVA) in the ovarian cortex of prepubertal (mean ±; SD; 93.0 ± 15.8 follicles/mm2) compared to adult (43.0 ± 13.6) females. Proportions of intact and viable follicles were similar (P>0.05) in fresh controls from prepubertal and adult ovarian tissues (intact follicles, 90.0 ± 5.8 and 92.5 ± 8.7%, respectively; viable follicles, 86.0 ± 2.7 and 82.5 ± 3.5%, respectively). Although cryopreservation decreased (P<0.05) the proportions of intact follicles compared to fresh controls, vitrification was superior (P<0.05) to slow freezing for preserving follicular structure in prepubertal (vitrification, 70.0 ± 3.4% intact follicles; slow freezing, 57.5 ± 8.7%) and adult ovarian tissues (vitrification, 73.5 ± 3.3%; slow freezing, 56.3 ± 4.8%). A similar pattern was observed for proportions of viable follicles, with vitrification exceeding (P<0.05) slow freezing in prepubertal (vitrification, 59.8 ± 4.6%; slow freezing, 37.8 ± 2.8%) and adult (vitrification, 62.5 ± 3.5%; slow freezing, 36.3 ± 4.5%) tissues. Interestingly, higher follicle densities in prepubertal versus adult ovaries had no influence (P>0.05) on follicle structure and viability post-thawing. Results demonstrate, for the first time, that vitrification permits retaining ≈80% of the original structure and ≈70% of the original viability of cat primordial follicles regardless of their density in the ovarian cortex. (Supported by the National Institutes of Health K01 RR020045).(platform)

Reproductive science as a discipline is making a difference in understanding and improving reproductive success in wildlife, including some of the world's most endangered species. Yet more than 97% of the world's 40,000 vertebrate species remain virtually unstudied in reproductive biology. Our laboratory's experiences over 25 years working worldwide have revealed almost as many mechanistic differences in reproduction as there are species. This basic biological information is important for demonstrating the remarkable diversity in how animals reproduce while warning us against "over-simplification" in our scientific field. Beyond scholarly knowledge, such data have practical use for implementing genetic management plans and for species recovery or even reintroduction into their natural habitat. Contributions can range from simply characterizing biological norms useful for improved breeding to the development of assisted reproductive technologies for propagating individuals located far from each other or refusing to naturally mate. The felids (cats) are particularly illustrative of the diversity of reproductive mechanisms among species from the same taxonomic family. The elephant and giant panda are excellent examples of the evolution of original ovulatory strategies, with the former having a double preovulatory LH surge and the latter a single ovulation per year. When adequate scholarly knowledge has been generated, there is a growing list of species whose recovery also is benefiting from reproductive science. For example, an increased understanding of basic reproductive physiology has allowed the ex situ giant panda population in China to more than double in the last decade. Combining intensive gamete/embryo studies with cryobiology are developing practical means for improving the intercontinental conservation and management of the cheetah population between Africa and the USA. In some cases, reproductive studies have been integrated with large-scale reintroduction efforts, examples including the scimitar-horned oryx and addax of North Africa, the Eld's deer of Southeast Asia and the black-footed ferret of North America. And such strategies are being expanded beyond charismatic vertebrates. One example includes exploring the mysteries of coral reproduction and how cryopreservation can play a role in species protection and recovery in ocean environments. In summary, we actually understand amazingly little about the reproductive physiology of whole animals. Besides illustrating the wonders of our discipline, such knowledge has high practical value for assisting in the recovery and, on occasion, restoration of species into nature.

The North American captive cheetah population is not self-sustaining due to poor reproductive efficiency. Of concern is that ∼25% of these females (∼132 total) are ≥9 years of age, and most have never reproduced. Our objectives were to assess (1) uterine morphology, (2) ovarian response to gonadotropins and (3) oocyte quality and fertilizability in cheetahs of different ages. We hypothesized that reproduction is compromised in aged cheetahs due to abnormal uterine physiology and reduced ovarian sensitivity and/or oocyte quality. Female cheetahs were divided into three age classes: young (2 to 5 y; n = 5), adult (6 to 8 y; n = 6) and aged (9 to 15 y; n = 6). Females received a single injection of 300 IU eCG followed 82–85 h later by an injection of 150 IU hCG. Twenty-six to 30 h post-hCG, uterine morphology was examined by ultrasonography, and follicle and corpora lutea numbers were determined by laparoscopy. Oocytes were recovered by aspiration, and a cohort from each female was immediately fixed for assessment of nuclear maturation. Remaining oocytes were inseminated in vitro with ∼1 × 106motile, frozen-thawed sperm/ml and cultured in Hams F10 with 5% fetal calf serum in 5% CO2in air. All embryos were fixed and stained (Hoechst 33342) to determine cell number. Data on ovarian and uterine morphology were analyzed by Proc-GLM procedures. Proportional data were pooled across age group and analyzed by non-parametric (NPAR1Way) procedures. Young females had a reduced (P < 0.05) uterine body wall thickness (3.1 ± 0.3 mm; mean ± SEM) compared to that for adult (4.6 ± 0.3 mm) or aged (4.4 ± 0.3 mm) females. However, there was no age effect (P > 0.05) on the entire width of the uterine body or the width or thickness of the uterine cornuae. Although there was a tendency for old females to produce fewer total follicles (≥2 mm) in response to exogenous gonadotropins, there was no statistical difference (P > 0.05) among groups (young, 19.4 ± 2.9; adult, 16.7 ± 2.7; aged, 10.5 ± 2.7). However, more (P < 0.05) oocytes were recovered from young (14.4 ± 3.0) and adult (17.7 ± 2.7) cheetahs compared to aged (7.3 ± 2.8) counterparts. Age had no effect (P > 0.05) on stage of oocyte maturation (germinal vesicle, metaphase I or metaphase II) at the time of aspiration or the proportion of mature oocytes that successfully fertilized in vitro in young (51.0 ± 16.0%), adult (64.4 ± 14.4%) and aged (83.3 ± 11.8%) females. There also was no influence (P > 0.05) of age on the proportion of fertilized oocytes that cleaved in vitro (55.6 ± 22.8 %, 38.1 ± 17.8% and 71.7 ± 12.3%). Overall, 26.2% of fertilized cheetah oocytes advanced to at least 8-cells in vitro with developmental success unaffected by age of donor. These data reveal a surprising lack of variation in female reproductive tract morphology and ovarian/gamete function among cheetahs of diverse ages. Although aged cheetahs produce fewer total oocytes, the quality of these gametes (on the basis of maturation, fertilization and development in vitro) is comparable to adult and young counterparts. Even though aged female cheetahs rarely naturally mate and conceive, our results suggest that recovered oocytes might be functional for producing embryos useful to contributing to genetic management. (Supported by the Ohrstrom Foundation, William H. Donner Foundation, Inc. and the Friends of the National Zoo). (platform)

Testicular sperm from the cat can be used via intracytoplasmic sperm injections (ICSI) to produce embryos in vitro, but the proportion of morulae and blastocysts is less than that obtained using ejaculated sperm. Compromised embryo development has been linked to the inability of the cat testicular sperm centrosome to form a normal sperm aster during the first cell cycle post-ICSI. The aim of the present study was to improve embryo development after ICSI with a testicular spermatozoon by centrosome/midpiece replacement from an ejaculated spermatozoon. Sperm suspensions used for ICSI (fresh testicular sperm from one adult testis vs. frozen-thawed sperm from one ejaculate in each replicate; four replicates) were sonicated for 3 s at setting 3 using a 60 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, USA) to separate heads from centrosome/midpieces. Control immunostaining with monoclonal centrin antibodies revealed that centrosomes were attached to midpieces and were separated from sperm heads after sonication. A single sperm head (testicular, Th; ejaculated, Eh) was carefully positioned proximal to a single centrosome/midpiece (testicular, Tc; ejaculated, Ec) before injection into an oocyte with a visible polar body (in each replicate, n = 18 oocytes injected with each ThEc, EhEc, EhTc, or ThTc combination; four replicates). Injected oocytes then were activated with 7% ethanol and cultured in vitro in Ham's F10 (38.5C, 5% CO2 in air). Percentages of first cleavages were recorded from 20 to 32 h post-activation (hpa). Nuclear status of uncleaved oocytes was evaluated at 48 hpa, and embryo development was assessed after 7 days of in vitro culture. Values were expressed as mean standard deviation and analyzed by ANOVA. None of the uncleaved oocytes were activated. Percentages of cleaved oocytes (relative to the total number of injected oocytes) were not different (range, 61-65%; P > 0.05) among combinations of sperm heads and centrosome/midpieces. The mean times of the first cleavage, however, were earlier (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 25.1 1.1 h; EhEc, 25.0 0.9 h) than with testicular centrosome/midpieces (EhTc, 29.2 0.8 h; ThTc, 29.3 1.2 h). Percentages of morulae produced were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 15.3 2.7%; EhEc, 16.7 2.1%) than with testicular centrosome/midpieces (EhTc, 8.3 3.1%; ThTc, 6.9 2.2%). Likewise, percentages of blastocysts were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 12.5 3.0%; EhEc, 13.9 2.8%) than with testicular centrosome/midpieces (EhTc, 5.6 3.3%; ThTc, 6.9 2.9%). These results demonstrated that: (1) kinetics of the first cell cycle and the success of embryonic development were determined by centrosome/midpiece source; and (2) poor developmental potential of domestic cat testicular sperm could be circumvented by centrosome/midpiece replacement from an ejaculated spermatozoon.

A thorough characterization of cryoprotectant (CPA) sensitivity is required to formulate a successful cryopreservation protocol for any biomaterial. The aim of this study was to characterize the toxic impact of various CPA types, concentrations, and exposure temperatures on the immature domestic cat oocyte. In Experiment 1, grade I immature oocytes (n=561) were exposed (30min; 25C or 0C) to 0M, 0.75M, 1.5M, or 3M of propylene glycol (PrOH) or ethylene glycol (EG) in PBS+20% fetal calf serum (v/v). After exposure, CPA was removed step-wise by subjecting oocytes to decreased CPA concentrations. Oocytes were cultured (30h; 38.5C, 5% CO2) in IVM medium as reported previously (Wolfe and Wildt 1996 J. Reprod. Fertil. 106, 135-141). Oocytes were then fixed and stained to examine nuclear status (Hoechst 33342) and spindle integrity (FITC-labeled anti--tubulin antibodies; Sigma Chemical Co., St. Louis, MO). Experiment 2 was designed on the basis of Experiment 1 results to assess the impact of the spindle abnormalities on subsequent embryo development. Oocytes (n=776) were exposed to CPA conditions yielding optimal nuclear maturation with either high (0.75M or 3.0M PrOH or 1.5M EG at 25C) or low (1.5M PrOH at 25C) proportions of abnormal spindle. After IVM, oocytes were inseminated with thawed semen (5105 motile sperm mL-1 ) in Ham's F-10 (Irvine Scientific, St-Anna, CA). At 16h post-insemination, oocytes were fixed and stained (Hoechst 33342) to assess IVF success (pronuclear formation) or cultured in vitro for 7 days to assess embryo development. Data were analyzed by ANOVA and Tukey's multiple comparison test. In Experiment 1, CPA treatment had no effect (NS) on meiotic progression to metaphase I. However, percentage of oocytes reaching metaphase II (MII) was reduced (P&lt;0.05) in 3.0M PrOH at 0C (29.38.3%; meanSD), 3.0M EG at 25C (33.78.9%), and 0C (29.411.0%) compared to all other conditions examined (range, 52.0% to 62.0%). All CPA treatments also increased (P&lt;0.05) spindle abnormalities at MII (range, 40.3% to 75.9%) compared to control (13.88.6%), except 1.5M PrOH at 25C (20.710.1%). None of the CPA treatments in Experiment 2 influenced IVF success (range, 55% to 63%; NS). However, percentage of cleaved embryos was reduced (P&lt;0.05) in 0.75M PrOH (32.14.1%), 1.5M EG (33.44.0%), and 3.0M PrOH (29.33.8%) compared to control (50.14.0%) or 1.5M PrOH (50.64.9%). Developmental competence (number of blastocysts relative to number of cleaved embryos) also was impaired (P&lt;0.05) in 1.5M EG (16.57.4%) and 3.0M PrOH (14.97.8%) compared to the other conditions (range, 32.5% to 38.5%), including 1.5 PrOH at 25C (32.57.8%). In conclusion, exposure of immature oocytes to 1.5M PrOH at 25C does not adversely impact oocyte maturation, MII spindle, fertilization, or embryo development in vitro in the domestic cat.

The Eld's deer, native to Southeast Asia, is threatened with extinction. Although artificial insemination is effective for offspring production, in vitro fertilization (IVF) would be more useful for rapidly disseminating genetic material from valuable founders. The objectives of this study were to: 1) determine if oocytes recovered from exogenous gonadotropin-treated hinds require additional in vitro maturation;; and 2) assess if fertilization is enhanced by supplementing Deer Synthetic Oviduct Fluid (DSOF;; Berg DK et al., 2003 Theriogenology 59, 189-205) with 1-day postestrus sheep serum (SS). Estrous cycles in Eld's deer hinds (n=10) were synchronized with PGF2 analog (Lutalyse", 500mg), followed by a 14-day intravaginal CIDR-G insertion;; ovine FSH (Ovagen"; 0.05 unit8 injections) was administered at 12-h intervals beginning 84h before CIDR-removal. COCs (n=160) were retrieved laparoscopically 40-46h post-CIDR-removal and either fixed or matured in vitro (for 12h v. 24h) in TCM-199 (Earle's salt) supplemented with 0.33mM pyruvate, 2mM glutamine, 100IUmL-1 penicillin, 100gmL-1 streptomycin, 10% fetal calf serum, 5gmL-1 FSH and LH and 1gmL-1 E2 (5% CO2, 38.5C). After 12- or 24-h IVM, cumulus cells were partially removed and oocytes (n=110) fertilized in DSOF with pooled frozen-thawed sperm (3 males;; 2106 motile sperm mL-1), in the absence or presence of SS (20%, v/v). Additional oocytes (n=18) were used for parthenogenetic control. At 20-h postinsemination, presumptive zygotes were fixed and stained (Hoechst 33342) to assess fertilization success (presence of two pronuclei). Data were analyzed by ANOVA. Overall, 16.02.6 (meanSEM) COCs were recovered/female. The majority of COCs were of excellent quality (grade I; 67.73.8%). At time of aspiration, 85% of the oocytes (n=11/13) were in metaphase I stage, 7.5% in telophase and 7.5% degenerate. No parthenogenic activation was observed. Likewise, no polyspermy was observed in any treatment. Fertilization was higher (P&lt;0.05) in oocytes matured for 24h and fertilized in the absence (64.43.1%) compared to presence (26.911.2%) of SS. In the absence of SS, a higher (P&lt;0.05) proportion of oocytes were fertilized after 24h (64.43.1%) compared to 12h (27.19.0%) IVM. There was no effect (P>0.05) of SS on fertilization among oocytes subjected to 12-h IVM (27.19.0% v. 12.59.5%). When SS was present during fertilization, no difference (P&lt;0.05) was observed among oocytes matured for 12 or 24h. Results demonstrate that: 1) Eld's deer oocytes require an additional 24-h IVM to complete maturation;; 2) DSOF supports sperm-oocyte interaction;; and 3) SS is not essential for successful fertilization. (Supported by Morris Animal Foundation.)