Abstract
Compared to somatic or sperm cells, mammalian oocytes are much more sensitive to cryopreservation mainly because of the large volume, cytoskeleton, and presence of the zona pellucida. ...Connections with the surrounding cumulus cells also are challenging to preserve in immature oocytes. Besides the genetic and epigenetic information enclosed in the nucleus, oocytes contain organelles and cytoplasmic factors that are necessary for the early embryo development. Therefore, exposure to cryoprotectant and freezing temperatures can easily damage the oocyte’s complex cellular structure and developmental competence, which leads to poor success in fertilization, lower embryo quality, and reduced pregnancy rates. Using the domestic cat as a model, our laboratory has studied damages in cumulus-oocyte complexes (immature oocytes) occurring at each step of the cryopreservation process. Specifically, this included the impact of cryoprotectants on microtubules, the effect of osmotic changes on the overall structures, as well as sensitivity of the nuclear chromatin and epigenetic patterns to freezing temperatures. Based on those findings, it has been possible to design mitigating solutions like customized ultra-rapid freezing or vitrification protocols, Laser-warming methods, or post-warming reanimations. New horizons have also been explored to move away from issues and limitations related to cryopreservation. Approaches like storage of oocytes for the long-term at non-freezing temperatures are currently being developed based on encouraging data generated in preliminary studies. For instance, incorporation of trehalose into the oocyte followed by microwave-assisted dehydration can allow to remove enough water while creating a protective trehalose glass compatible with survival and storage at non-freezing temperatures. Interestingly, transcriptomic and proteomic studies have already shown that stresses and damages induced by dehydration versus cryopreservation are different and may be easier to overcome.
Studying the reproductive biology of wild animal species produces knowledge beneficial to their management and conservation. However, wild species also share intriguing similarities in reproductive ...biology with humans, thereby offering alternative models for better understanding the etiology of infertility and developing innovative treatments. The purpose of this review is to raise awareness in different scientific communities about intriguing connections between wild animals and humans regarding infertility syndromes or improvement of fertility preservation. The objectives are to (1) highlight commonalities between wild species and human fertility, (2) demonstrate that research in wild species-assisted reproductive technologies can greatly enhance success in human reproductive medicine, and (3) recognize that human fertility preservation is highly inspiring and relevant to wild species conservation. In addition to having similar biological traits in some wild species and humans, the fact of sharing the same natural environment and the common needs for more options in fertility preservation are strong incentives to build more bridges that will eventually benefit both animal conservation and human reproductive medicine.
The North American cheetah (Acinonyx jubatus) population serves as both an insurance population for their rapidly decreasing wild cohorts as well as a research population to understand the unique ...biology of this species. This review focus on the complexity of the female cheetah reproductive system and the recent advances that have been made towards understanding basic biology and reproductive function, and application of assisted breeding technologies to enhance reproduction and maintain genetic diversity of this species in human care. Cheetah females are non-seasonal breeders that exhibit lengthy periods of anestrus that are not associated with age, environment, or reproductive potential. It is possible to collect good quality oocytes, that support fertilisation and successful early embryonic development, regardless of female age (from 2 to 12 yr old). However, the prevalence of uterine pathologies increases with age and prevents middle to advanced age females from establishing pregnancy. Pregnancy can be diagnosed in non-sedated cheetah females via ultrasonography (first month), steroid hormone analysis (second/third month) or radiography (third month). Fecal biomarkers, such as Immunoglobulin J, show great promise for diagnosing pregnancy at an early stage as well as other physiological states. Several decades of basic research have led to efficient management of natural breeding and recent successes in assisted reproduction.
•Cheetah females are non-seasonal breeders and exhibit lengthy periods of anestrus that are not associated with age, environment, or reproductive potential.•Good quality oocytes, that support fertilisation and successful early embryonic development, can be harvested from females regardless of age (from 2 to 12 yr old).•Pregnancy can be diagnosed in cheetah females via ultrasonography (first trimester), steroid hormone analysis (second/third) or radiography (third trimester).•Fecal biomarkers, such as Immunoglobulin J, show promise for diagnosing pregnancy at an early stage as well as other physiological states.
Many parts of the animal and human body host groups of bacteria, viruses, and fungi that together are known as the microbiome. Microbiomes do not cause disease but are important for the healthy ...working of many systems in the body, including for reproduction and fertility. While the microbiome that lives in a reproductive tract play the most direct role, microbiomes from other areas of the body may also affect reproductive health. However, not much is known about how these groups of microorganisms regulate fertility as well as the health of parents and offspring and help animals to cope with environmental changes. Furthermore, compared to the large amount of research in laboratory species and humans, there is less information about domestic or wild animal species. This special series of Reproduction and Fertility on microbiomes is aimed at filling this gap with articles from experts highlighting important evidence in reproductive microbiomes, current research gaps, and new directions.
Contents
Ovarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to ...investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra‐ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1 × 1 × 0.2 mm3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions (VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24 hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7 days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10 min had the highest percentage of normal follicles (approximately 20% after 7 days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification.
A major challenge to retaining viability of frozen gametes and reproductive tissues is to understand and overcome species-specificities, especially because there is substantial diversity in ...cryobiological properties and requirements among cell types and tissues. Systematic studies can lead to successful post-thaw recovery, especially after determining: 1) membrane permeability to water and cryoprotectant, 2) cryoprotectant toxicity, 3) tolerance to osmotic changes, and 4) resistance to cooling and freezing temperatures. Although species-dependency ultimately dictates the ability of specific cells and tissues to survive freeze-thawing, there are commonalities between taxa that allow a protocol developed for one species to be useful information for another. This is the reason for performing comparative cryopreservation studies among diverse species. Our laboratory has compared cellular cryotolerance, especially in spermatozoa, in a diverse group of animals—from corals to elephants—for more than 30 yrs. Characterizing the biophysical traits of gametes and tissues is the most efficient way to develop successful storage and recovery protocols, but, such data are only available for a few laboratory, livestock, and fish species, with virtually all others (wild mammals, birds, reptiles, and amphibians) having gone unstudied. Nonetheless, when a rare animal unexpectedly dies, there is no time to understand the fundamentals of biophysics. In these emergencies, it is necessary to rely on experience and the best data from taxonomically-related species. Fortunately, there are some general similarities among most species, which, for example, allow adequate post-thaw viability. Regardless, there is a priority for more information on biophysical traits and freezing tolerance of distinctive biomaterials, especially for oocytes and gonadal tissues, and even for common, domesticated animals. Our colleague, Dr John Critser was a pioneer in cryobiology, earning that moniker because of his advocacy and devotion to understanding the differences (and similarities) among species to better store living genetic material.
Abstract
STUDY QUESTION
Do nuclear proteins in the germinal vesicle (GV) contribute to oocyte competence acquisition during folliculogenesis?
SUMMARY ANSWER
Proteomic analysis of GVs identified ...candidate proteins for oocyte competence acquisition, including a key RNA processing protein–heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1).
WHAT IS KNOWN ALREADY
The domestic cat GV, which is physiologically similar to the human GV, gains the intrinsic ability to resume meiosis and support early embryo development during the pre-antral-to-antral follicle transition. However, little is known about nuclear proteins that contribute to this developmental process.
STUDY DESIGN SIZE, DURATION
GVs were enriched from pre-antral (incompetent) and antral (competent) follicles from 802 cat ovaries. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in GVs from the two follicular categories.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Two biological replicates (from independent pools of ovaries) of pre-antral versus antral samples were labeled by tandem mass tags and then assessed by liquid chromatography–tandem mass spectrometry. Proteomic data were analyzed according to gene ontology and a protein–protein interaction network. Immunofluorescent staining and protein inhibition assays were used for validation.
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 174 nuclear proteins was identified, with 54 being up-regulated and 22 down-regulated (≥1.5-fold) after antrum formation. Functional protein analysis through gene ontology over-representation tests revealed that changes in molecular network within the GVs during this transitional phase were related to chromatin reorganization, gene transcription, and maternal RNA processing and storage. Protein inhibition assays verified that hnRNPA2B1, a key nuclear protein identified, was required for oocyte meiotic maturation and subsequent blastocyst formation.
LARGE SCALE DATA
Data are available via ProteomeXchange with identifier PXD007211.
LIMITATIONS REASONS FOR CAUTION
Proteins identified by proteomic comparison may (i) be involved in processes other than competence acquisition during the pre-antral-to-antral transition or (ii) be co-expressed in other macrostructures besides the GV. Expressional and functional validations should be performed for candidate proteins before downstream application.
WIDER IMPLICATIONS OF THE FINDINGS
Collective results generated a blueprint to better understand the molecular mechanisms involved in GV competence acquisition and identified potential nuclear competence markers for human fertility preservation.
STUDY FUNDING AND COMPETING INTEREST(S)
Funded by the National Center for Research Resources (R01 RR026064), a component of the National Institutes of Health (NIH) and currently by the Office of Research Infrastructure Programs/Office of the Director (R01 OD010948). The authors declare that there is no conflict of interest.
Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. ...Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%–1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing technique with glycerol as a principle cryoprotectant is simplified and applicable for cat testicular tissue cryopreservation. We also demonstrate for the first time that feline spermatozoa derived from frozen-thawed testicular tissues retain their fertilizing ability and can be used to produce ICSI-derived embryos.
BACKGROUND
Chromatin configuration of the germinal vesicle (GV) and quality of the cytoplasm are critical factors in achieving oocyte meiotic and developmental capacity during folliculogenesis. ...Besides gaining new insights into the timing and cellular mechanisms associated with the acquisition and regulation of GV oocyte competence, the domestic cat model was used to examine (i) the relation between GV chromatin configuration and oocyte functionality during folliculogenesis and (ii) the role of the cytoplasmic environment on the GV competence and stability.
METHODS
Structural and functional properties of GV oocytes were characterized after isolation from different follicle stages of non-stimulated cat ovaries. GV transfers, artificial chromatin compaction and oocyte vitrification were used to demonstrate the respective roles of GV and cytoplasm on the oocyte functionality.
RESULTS
GVs acquired the intrinsic capability to resume meiosis during the pre-antral follicle stage, whereas the capacity to support embryo development occurred while the antrum started to form. Chromatin configuration of the GV did not undergo extensive modification during the acquisition of competence or during the arrest of transcriptional activity at the large antral follicle stage. However, the quality and quantity of the cytoplasm regulated and enhanced GV functionality. This finding also held for GVs transferred from incompetent or subpar oocytes into the cytoplasm of good quality oocytes or when chromatin was artificially modified or vitrified.
CONCLUSIONS
The cat model provides a new insight into GV oocyte structure and function during folliculogenesis while challenging current concepts about oocyte quality criteria based on the GV morphology. This suggests alternative evaluative approaches for oocytes from other species too, including humans. Cat GVs also appear competent at an early follicle stage and are resilient to perturbations which designate this organelle as an attractive target for developing novel fertility preservation tactics.
The objectives of the present study were to evaluate sperm characteristic of captive clouded leopards in Thailand and examine the structural and functional properties of sperm after selection with ...the single-layer centrifugation (SLC) method. Twenty-two ejaculates from 11 captive clouded leopards (four housed with access to a female in estrus, and seven housed singly) were collected and assessed for semen traits during 2013 to 2015. Twelve fresh ejaculates were chosen from seven males, and each was divided between two sperm preparation methods; (1) simple washing and (2) SLC. Cryopreservation was performed after semen preparation. Sperm qualities after selections including motility, progressive motility, sperm motility index, viability, acrosome integrity, DNA integrity, and morphology were evaluated in fresh, chilled, and frozen-thawed samples. In addition, sperm functionality after cryopreservation was tested by heterologous IVF using domestic cat oocytes. Sperm motility in the ejaculates was 52.5% to 91.3% (76.8 ± 2.0%, mean ± standard error). A high proportion of morphologically abnormal sperm (63.9 ± 2.0%) was observed, with the major abnormality being tightly coiled tail (13.5 ± 0.5%). An interesting observation was that males housed together with a female had a significantly higher proportion of sperm with intact acrosome (47.9 ± 3.4% and 38.4 ± 2.8%) and lower proportion of sperm with bent midpiece and droplet (7.1 ± 0.6% and 10.2 ± 0.5%) than the males living singly. The sperm motility index, intact acrosome, and sperm with normal tail in the fresh and chilled semen samples were improved by the SLC. In the postthawed semen, the SLC selected higher numbers of viable sperm (34.1 ± 2.2% and 27.9 ± 1.8%), sperm with intact acrosome (31.2 ± 2.1% and 24.3 ± 2.2%), and sperm with normal tail (34.2 ± 2.7% and 24.3 ± 2.7%) than simple washing. Also, the proportion of sperm with tightly coiled tail was lower in the SLC-processed than the simple washed samples (8.1 ± 3.1% and 13.5 ± 3.4%). The SLC-processed group had significantly higher penetration rate in heterologous IVF (29.4 ± 3.0%) than the simple washing group (15.8 ± 3.2%). In conclusion, ejaculates of clouded leopards living in Thailand demonstrated teratospermic characteristic similar to the previous reports from other continents. Single-layer centrifugation is a promising tool to select morphologically normal sperm of teratospermic donors. The successes of assisted reproductive technology could be enhanced by the improved quality of postthaw sperm in this species.
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