Pierre Comizzoli

Lessons from biodiversity - the value of nontraditional species to advance reproductive science, conservation, and human health.

Reproduction is quintessential to species survival. But what is underappreciated for this discipline is the wondrous array of reproductive mechanisms among species— variations as diverse as the morphology of the species themselves (more than 55,000 vertebrate and 1.1 million invertebrate types). We have investigated only a tiny fraction of these species in reproductive science. Besides the need to fill enormous gaps in a scholarly database, this knowledge has value for recovering and genetically managing rare species as well as addressing certain reproductive issues in humans. This article provides examples, first to advise against oversimplifying reproduction and then to show how such knowledge can have practical use for managing whole animals, populations, or even saving an entire species. We also address the expected challenges and opportunities that could lead to creative shifts in philosophy and effective actions to benefit more species as well as a future generation of reproductive scientists. Mol. Reprod. Dev. 77: 397–409, 2010.

Effect of 1,2-Propanediol Versus 1,2-Ethanediol on Subsequent Oocyte Maturation, Spindle Integrity, Fertilization, and Embryo Development In Vitro in the Domestic Cat

Abstract This study assessed the impact of various cryoprotectant (CPA) exposures on nuclear and cytoplasmic maturation in the immature cat oocyte as a prerequisite to formulating a successful cryopreservation protocol. In experiment 1, immature oocytes were exposed to 0, 0.75, 1.5, or 3.0 M of 1,2-propanediol (PrOH) or 1,2-ethanediol (EG) at room temperature (25°C) or 0°C for 30 min. After CPA removal and in vitro maturation, percentage of oocytes reaching metaphase II (MII) was reduced after exposure to 3.0 M PrOH at 0°C or 3.0 M EG at both temperatures. All CPA exposures increased MII spindle abnormalities compared to control, except 1.5 M PrOH at 25°C. In experiments 2 and 3, immature oocytes were exposed to CPA conditions yielding optimal nuclear maturation that either had caused spindle damage (0.75 M PrOH, 1.5 M EG, and 3.0 M PrOH at 25°C) or not (1.5 M PrOH at 25°C). After maturation and insemination in vitro, oocytes were cultured for 7 days to assess treatment influence on developmental competence. CPA exposure did not affect fertilization, but the high incidence of MII spindle abnormalities resulted in a low percentage of cleaved embryos. Blastocyst formation and quality were influenced by both CPA types (EG was more detrimental than PrOH) and concentration (3.0 M was more detrimental than 1.5 M). Overall, cat oocytes appear to be highly sensitive to CPA except after exposure to 1.5 M PrOH at 25°C, a treatment that still allowed ∼60% of the oocytes to reach MII and ∼20% to form blastocysts.

Protecting and Extending Fertility for Females of Wild and Endangered Mammals

Sustaining viable populations of any wildlife species requires a combination of adequate habitat protection and 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 nature). Assisted reproductive technologies developed for addressing human infertility and enhancing livestock production have shown encouraging promise in a few 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 endangered species numbers. 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 components of the rapidly emerging field of oncofertility in women that are highly compatible with preserving valuable genomes of individuals or populations of threatened wildlife. Strategies associated with ovarian tissue cryopreservation and follicle in vitro culture are especially attractive for protecting and extending fertility for wild females. Given adequate attention and more basic studies, we predict that these approaches could assist in the intensive and practical management of gene diversity in endangered species.

Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species

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.

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

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.

Increasing Age Influences Uterine Integrity, But Not Ovarian Function or Oocyte Quality, in the Cheetah (Acinonyx jubatus)

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.

Poor Centrosomal Function of Cat Testicular Spermatozoa Impairs Embryo Development In Vitro after Intracytoplasmic Sperm Injection

Abstract 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.

Paracrine factors from cumulus-enclosed oocytes ensure the successful maturation and fertilization in vitro of denuded oocytes in the cat model

OBJECTIVE To better characterize cumulus-oocyte interactions during oocyte maturation and fertilization in the cat model. DESIGN Experimental in vitro study. SETTING Smithsonian Institution. ANIMAL(S) Domestic shorthair cats. INTERVENTION(S) Groups of denuded oocytes (DOs) and cumulus-oocyte complexes (COCs) were subjected to in vitro maturation (with or without FSH and LH, with or without the gap junction disruptor 1-heptanol, in separated groups or in coculture) and inseminated in vitro (IVF; in separated groups or in coculture). MAIN OUTCOME MEASURE(S) Nuclear maturation, pronuclear formation, kinetics of early embryo cleavage, and blastocyst formation and quality after different in vitro conditions were compared between DOs cultured separately and DOs cocultured with COCs. RESULT(S) Without FSH and LH, the removal of cumulus cells prevented spontaneous meiotic resumption in DOs. With FSH and LH, groups of DOs progressed to the metaphase I stage but fully advanced to metaphase II only in coculture with intact (nondisrupted) COCs. Groups of DOs cultured separately were fertilized poorly and exhibited no blastocyst formation. In contrast, DOs cocultured with intact COCs during in vitro maturation and IVF recovered fertilizability, and approximately 35% formed blastocysts. CONCLUSION(S) Paracrine factors produced by cumulus-enclosed oocytes in the cat model will help to develop synthetic media for successful in vitro culture of DOs.

In Vitro Compaction of Germinal Vesicle Chromatin is Beneficial to Survival of Vitrified Cat Oocytes

The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8-16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes.

Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.

The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P < 0.05). Morphologically normal primordial follicles were higher in the sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P < 0.05). Moreover, less apoptotic primordial follicles were found in both sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P < 0.05). However, their Cx 43 expression showed no difference among the groups of different sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P < 0.05). In addition, the slow freezing resulted in more postthawed morphologically normal primordial follicles (38.8% vs. 28.3%, P < 0.05) and less apoptotic primordial follicles (3.8% vs. 8.9%, P < 0.05) than vitrification. The Cx 43 expression showed no difference between slow freezing and vitrification. The present study reported the positive effects of sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.