Microwave-assisted dehydration, long-term storage at non-freezing temperatures, and rehydration of cat germinal vesicles.

Germinal vesicles (GVs) are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved GVs can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a GV 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 GVs. 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 GV oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates 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.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

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, GM1 localization 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.

Modeling genetic benefits and financial costs of integrating biobanking into the conservation breeding of managed marsupials.

Managed breeding programs are an important tool in marsupial conservation efforts but may be costly and have adverse genetic effects in unavoidably small captive colonies. Biobanking and assisted reproductive technologies (ARTs) could help overcome these challenges, but further demonstration of their potential is required to improve uptake. We used genetic and economic models to examine whether supplementing hypothetical captive populations of dibblers (Parantechinus apicalis) and numbats (Myrmecobius fasciatus) with biobanked founder sperm through ARTs could reduce inbreeding, lower required colony sizes, and reduce program costs. We also asked practitioners of the black-footed ferret (Mustela nigripes) captive recovery program to complete a questionnaire to examine the resources and model species research pathways required to develop an optimized biobanking protocol in the black-footed ferret. We used data from this questionnaire to devise similar costed research pathways for Australian marsupials. With biobanking and assisted reproduction, inbreeding was reduced on average by between 80% and 98%, colony sizes were on average 99% smaller, and program costs were 69- to 83-fold lower. Integrating biobanking made long-standing captive genetic retention targets possible in marsupials (90% source population heterozygosity for a minimum of 100 years) within realistic cost frameworks. Lessons from the use of biobanking technology that contributed to the recovery of the black-footed ferret include the importance of adequate research funding (US$4.2 million), extensive partnerships that provide access to facilities and equipment, colony animals, appropriate research model species, and professional and technical staff required to address knowledge gaps to deliver an optimized biobanking protocol. Applied research investment of A$133 million across marsupial research pathways could deliver biobanking protocols for 15 of Australia's most at-risk marsupial species and 7 model species. The technical expertise and ex situ facilities exist to emulate the success of the black-footed ferret recovery program in threatened marsupials using these research pathways. All that is needed now for significant and cost-effective conservation gains is greater investment by policy makers in marsupial ARTs.

Los programas de reproducción controlada son una herramienta importante para los esfuerzos de conservación de marsupiales, aunque pueden resultar costosos y tener efectos genéticos adversos en las colonias cautivas incapaces de aumentar en tamaño. Los biobancos y las tecnologías de reproducción asistida (TRA) podrían ayudar a superar estos problemas, pero es necesario seguir demostrando su potencial para mejorar su adopción. Utilizamos modelos genéticos y económicos para analizar si la introducción de esperma fundador proveniente de biobancos mediante tecnologías de reproducción asistida a poblaciones cautivas hipotéticas de los marsupiales Parantechinus apicalis y Myrmecobius fasciatus podría reducir la endogamia, disminuir el tamaño efectivo de las colonias y reducir el costo de los programas. También pedimos a los profesionales del programa de recuperación en cautiverio del hurón de patas negras (Mustella nigripes) que respondieran un cuestionario para analizar los recursos y los métodos de investigación de las especies modelo necesarias para desarrollar un protocolo de biobanco optimizado para el hurón de patas negras. Utilizamos los datos de este cuestionario para diseñar métodos de investigación con costos similares para los marsupiales australianos. Con el biobanco y la reproducción asistida, la endogamia se redujo en promedio entre un 80 y un 98%, el tamaño de las colonias fue en promedio un 99% más pequeño y los costos del programa entre 69 y 83 veces menores. La integración del biobanco posibilitó los objetivos de retención genética en cautiverio a largo plazo en marsupiales (90% de heterocigosidad de la población de origen durante un mínimo de 100 años) dentro de un marco realista de costos. Entre el aprendizaje extraído del uso de la tecnología de biobancos que contribuyó a la recuperación del hurón de patas negras figuran la importancia de una financiación adecuada de la investigación (4.2 millones de dólares), colaboraciones profundas que faciliten el acceso a instalaciones y equipos, colonias de animales, especies modelo adecuadas para la investigación y el personal profesional y técnico necesario para abordar las lagunas de conocimiento y ofrecer un protocolo optimizado para los biobancos. Una inversión en investigación aplicada de 133 millones de dólares australianos para la investigación de los marsupiales podría proporcionar protocolos de biobancos para 15 de las especies de marsupiales australianos en mayor riesgo y 7 especies modelo. Existen los conocimientos técnicos y las instalaciones ex situ para emular el éxito del programa de recuperación del hurón de patas negras en marsupiales amenazados utilizando estas vías de investigación. Ahora sólo se necesita una mayor inversión por parte de los responsables políticos de las TRA para marsupiales para obtener beneficios de conservación significativos y rentables.

Gut bacterial composition shows sex-specific shifts during breeding season in ex situ managed black-footed ferrets.

The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus - all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets.

Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying.

PURPOSE: Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration. METHODS: Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR. RESULTS: CRNPs effectively transported trehalose into COCs within 4 h of co-incubation at 38.5 °C followed by a cold-triggered release at 4 °C for 15 min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1 at dehydration levels reaching up to 63% of water loss. CONCLUSION: Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

Transfer of Galectin-3-Binding Protein via Epididymal Extracellular Vesicles Promotes Sperm Fertilizing Ability and Developmental Potential in the Domestic Cat Model.

Key proteins transferred by epididymal extracellular vesicles (EVs) to the transiting sperm cells contribute to their centrosomal maturation and developmental potential. Although not reported in sperm cells yet, galectin-3-binding protein (LGALS3BP) is known to regulate centrosomal functions in somatic cells. Using the domestic cat model, the objectives of this study were to (1) detect the presence and characterize the transfer of LGALS3BP via EVs between the epididymis and the maturing sperm cells and (2) demonstrate the impact of LGALS3BP transfer on sperm fertilizing ability and developmental potential. Testicular tissues, epididymides, EVs, and spermatozoa were isolated from adult individuals. For the first time, this protein was detected in EVs secreted by the epididymal epithelium. The percentage of spermatozoa with LGALS3BP in the centrosome region increased as cells progressively incorporated EVs during the epididymal transit. When LGALS3BP was inhibited during in vitro fertilization with mature sperm cells, less fertilized oocytes and slower first cell cycles were observed. When the protein was inhibited in epididymal EVs prior to incubation with sperm cells, poor fertilization success further demonstrated the role of EVs in the transfer of LGALS3BP to the spermatozoa. The key roles of this protein could lead to new approaches to enhance or control fertility in clinical settings.

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation.

Long-term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long-term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long-term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.

Novel Proteomic Profiling of Epididymal Extracellular Vesicles in the Domestic Cat Reveals Proteins Related to Sequential Sperm Maturation with Differences Observed between Normospermic and Teratospermic Individuals.

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, 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. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.

Long-term storage of gametes and gonadal tissues at room temperatures: the end of the ice age?

Long-term preservation of viable spermatozoa, eggs, embryos, and gonadal tissues of good quality is essential in human reproductive medicine and for the population management of livestock, laboratory, and wild species. Instead of using freezing temperatures, encouraging findings indicate that structures and functions of gametes or gonadal tissues can be suspended in trehalose glass after dehydration and then preserved at supra-zero temperatures. As a new era in fertility preservation and biobanking is about to start, the advantages, needs, and implications of germplasm storage at room temperatures must be carefully examined. Although very promising, the development of alternate biobanking strategies does not necessarily mean that the end of the "ice age" (cryopreservation) is near.

Desiccated cat spermatozoa retain DNA integrity and developmental potential after prolonged storage and shipping at non-cryogenic temperatures.

PURPOSE: To evaluate the DNA integrity and developmental potential of microwave-dehydrated cat spermatozoa after storage at - 20 °C for different time periods and/or overnight shipping on dry ice. METHODS: Epididymal spermatozoa from domestic cats were microwave-dehydrated on coverslips after trehalose exposure. Dried samples were either assessed immediately, stored for various duration at - 20 °C, or shipped internationally on dry ice before continued storage. Dry-stored spermatozoa were rehydrated before assessing DNA integrity (TUNEL assays) or developmental potential (injection into in vitro matured oocytes followed by in vitro embryo culture for up to 7 days). RESULTS: Percentages of dried-rehydrated spermatozoa with intact DNA was not significantly affected (P > 0.05) by desiccation and short-term storage (range, 78.9 to 80.0%) but decreased (P < 0.05) with storage over 5 months (range, 71.0 to 75.2%) compared to fresh controls (92.6 ± 2.2%). After oocyte injection with fresh or dried-rehydrated spermatozoa (regardless of storage time), percentages of activation, pronuclear formation, and embryo development were similar (P > 0.05). Importantly, spermatozoa shipped internationally also retained the ability to support embryo development up to the morula stage. CONCLUSION: Results demonstrated the possibility to sustain DNA integrity and developmental potential of spermatozoa by dry-preservation, even after long-term storage and long-distance shipment at non-cryogenic temperatures. While further studies are warranted, present results demonstrate that dry preservation can be a reliable approach for simple and cost-effective sperm biobanking or shipment.

Transcriptome dynamics in developing testes of domestic cats and impact of age on tissue resilience to cryopreservation.

BACKGROUND: Fundamental knowledge of cellular and molecular mechanisms in developing testicular tissues is critical to better understand gonadal biology and responses to non-physiological conditions. The objective of our study was to (1) analyze transcriptome dynamics in developing testis of the domestic cat and (2) characterize age effects on the initial response of the tissue to vitrification. Tissues from adult and juvenile cats were processed for histology, DNA integrity, and RNA sequencing analyses before and after vitrification. RESULTS: Transcriptomic findings enabled to further characterize juvenile period, distinguishing between early and late juvenile tissues. Changes in gene expression and functional pathways were extensive from early to late juvenile to adult development stages. Additionally, tissues from juvenile animals were more resilient to vitrification compared to adult counterparts, with early juvenile sample responding the least to vitrification and late juvenile sample response being closest to adult tissues. CONCLUSIONS: This is the first study reporting comprehensive datasets on transcriptomic dynamic coupled with structural analysis of the cat testis according to the age and exposure to cryopreservation. It provides a comprehensive network of functional terms and pathways that are affected by age in the domestic cat and are either enriched in adult or juvenile testicular tissues.

Endometrial receptivity and embryo implantation in carnivores-commonalities and differences with other mammalian species.

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.

Adding new ingredients to the recipe for successful embryo transfers.

The success of a pregnancy and the birth of a heathy baby following embryo transfer are conditioned by many factors, including embryo quality and the uterine environment. While we keep looking for more indicators of embryo quality, it also is critical to understand what constitutes a favorable uterine environment leading to a successful pregnancy and birth. This issue of JARG offers new insights on both components-so called by some "the seed and the soil"-and also highlights the critical interactions between the two. Collectively, these publications are contributing to a better understanding of basic embryology and reproductive biology. They could lead to multiple applications to mitigate infertility issues; however, our knowledge base remains rudimentary when it comes to sorting out the 'soil or seed' dilemma. The call from all authors for more research in their respective areas resonates within the ART community. Recognizing the practical and ethical limitations of studies in human patients also reemphasizes the need for solid research in multiple animal models to better understand what constitutes the best recipe for successful embryo transfer outcomes.

Exposure to epididymal extracellular vesicles enhances immature sperm function and sustains vitality of cryopreserved spermatozoa in the domestic cat model.

PURPOSE: Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer key factors to maturing spermatozoa. Using an in vitro system previously developed in our laboratory, the objective was to (1) characterize the impact of EV exposure on the fertilizing ability and developmental potential of immature sperm cells from the caput epididymidis and (2) examine the benefit of EV exposure to restore vitality of mature spermatozoa from the cauda epididymidis after freezing-thawing. METHODS: EVs were isolated from entire epididymides and collected into pellets via ultracentrifugation. Immature spermatozoa from adult cats were isolated from the caput epididymis and incubated with EVs prior to in vitro fertilization. Similarly, mature spermatozoa were isolated from the cauda segment and cryopreserved prior to EV exposure and subsequent analysis of motility and developmental potential after fertilization. RESULTS: EV exposure did not affect the percentage of caput sperm penetration; however, it improved the fertilizing ability (faster pronuclear apposition) and the developmental potential (higher proportions of morula-blastocysts) of those immature sperm cells. While EV exposure was beneficial to the frozen-thawed sperm motility, it did not significantly improve the fertilizing ability and the developmental potential. CONCLUSIONS: Epididymal EVs contain multiple factors contributing to immature sperm function, specifically enhancing the ability to complete a faster pronuclear apposition with subsequently improved early embryonic development. Supplementation was also beneficial to the motility of spermatozoa that had undergone cryopreservation. Those new findings could lead to new options for male fertility treatment in animal models and humans.

Initial response of ovarian tissue transcriptome to vitrification or microwave-assisted dehydration in the domestic cat model.

BACKGROUND: Long term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Compared to single cell preservation, optimization of protocols for tissues is highly complex because of the diversity of cells responding differently to non-physiological conditions. Using the prepubertal domestic cat as a model, the objective was to study immediate effects of vitrification or microwave-assisted dehydration on the global transcriptome dynamics in the ovarian cortex. RNA sequencing was performed on ovarian tissues (n = 6 individuals) from different conditions: fresh tissue after dissection (F), vitrified/warmed tissue (V), tissue dehydrated for 5 min (D5) or 10 min (D10) followed by rehydration. Differential gene expression analysis was performed for comparison pairs V vs. F, D10 vs. F, D5 vs. F and D10 vs. D5, and networks were built based on results of functional enrichment and in silico protein-protein interactions. RESULTS: The impact of the vitrification protocol was already measurable within 20 min after warming and involved upregulation of the expression of seven mitochondrial DNA genes related to mitochondrial respiration. The analysis of D10 vs. F revealed, 30 min after rehydration, major downregulation of gene expression with enrichment of in silico interacting genes in Ras, Rap1, PI3K-Akt and MAPK signaling pathways. However, comparison of D5 vs. F showed negligible effects of the shorter dehydration protocol with two genes enriched in Ras signaling. Comparison of D10 vs. D5 showed downregulation of only seven genes. Vitrification and dehydration protocols mainly changed the expression of different genes and functional terms, but some of the differentially expressed genes formed a major in silico protein-protein interaction cluster enriched for mitochondrial respiration and Ras/MAPK signaling pathways. CONCLUSIONS: Our results showed, for the first time, different effects of vitrification and microwave-assisted dehydration protocols on the global transcriptome of the ovarian cortex (using the domestic cat as a biomedical model). Acquired data and networks built on the basis of differentially expressed genes (1) can help to better understand stress responses to non-physiological stresses and (2) can be used as directions for future preservation protocol optimizations.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca).

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ±â€¯7.7%) and membrane functionality (60.5 ±â€¯4.2%) and higher mitochondrial activity (21.5 ±â€¯3.7%) than those preserved in ACP-117c (20.9 ±â€¯5.4% motile sperm; 47.1 ±â€¯2.5% functional membrane; 11.8 ±â€¯1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ±â€¯3.3%) in comparison to samples diluted in ACP-117c (18.6 ±â€¯1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

Breakthroughs and new horizons in reproductive biology of rare and endangered animal species.

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.

Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†.

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.

Desiccation and supra-zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation.

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long-term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes' germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming.

Endogenous pluripotent factor expression after reprogramming cat fetal fibroblasts using inducible transcription factors.

Incomplete transgene-silencing remains a challenge in the generation of induced pluripotent stem cells (iPSC) in felids-a critical family in biomedical and biodiversity conservation science. In this study doxycycline-inducible transgenes (NANOG, POU5F1, SOX2, KLF4, and cMYC) were used to reprogram cat fetal fibroblasts with the objective of obtaining iPSC with fully silenced transgenes. Colony formation was slower (14 vs. 8 days) and at lower efficiency than mouse embryonic fibroblasts (0.002% vs. 0.02% of seeded cells). Alkaline-phosphatase positive colonies were grown on feeder cells plus LIF and GSK3, MEK, and ROCK inhibitors. Cells could be passaged singly and transgene expression was silenced at passage 3 (P3) after doxycycline removal at P2. NANOG, POU5F1, and SOX2 were expressed at P3, P6, and P10, although at lower immunostaining intensities than in cat inner cell masses (ICM). Transcripts related to pluripotency (NANOG, POU5F1, SOX2, KLF4, cMYC, and REX1) and differentiation (FGF5, TBXT, GATA6, SOX17, FOXF1, PAX6, and SOX1) were assessed by a reverse transcription-quantitative polymerase chain reaction in iPSC and embryoid bodies. The immunostaining patterns, relatively low levels of NANOG and REX1 in comparison to ICM along with the expression of TBXT (mesoderm) suggested that cells were a mix of reprogrammed pluripotent and differentiating cells.