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.

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.

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.

Key factors enhancing sperm fertilizing ability are transferred from the epididymis to the spermatozoa via epididymosomes in the domestic cat model.

PURPOSE: Spermatozoa undergo critical changes in structure and function during the epididymal transit. Our previous studies in the domestic cat demonstrated that incidence of cenexin-a key protein involved in the centrosomal maturation-progressively increases in sperm cells from caput to cauda epididymidis. The objectives of the study were to (1) characterize mechanisms involved in transferring key factors-using the cenexin as a marker-between the epididymis and maturing sperm cells and (2) demonstrate the impact of such mechanisms on the acquisition of functional properties by spermatozoa. METHODS: Epididymides were dissected from adult cat testes to assess the presence and localization of cenexin in testicular tissues and each epididymal segment (caput, corpus, and cauda) via immunofluorescence, Western blot, and mass spectrometry. RESULTS: Results showed that tissues, luminal fluid, and isolated epididymosomes from each segment contained cenexin. Co-incubation of immature sperm cells for 3 h with luminal fluid or epididymosomes followed by immunostaining revealed that percentages of sperm cells containing cenexin significantly increased in samples co-incubated with epididymosome suspensions. Additionally, epididymosome co-incubation with immature spermatozoa resulted in sustained motility compared to untreated spermatozoa while there was no significant effect on acrosome integrity. CONCLUSIONS: Taken together, these results suggest that epididymosomes play a critical role in epididymal sperm maturation and could be ideal vehicles to assist in the enhancement or suppression of male fertility.

Current knowledge in the biology of gametes and embryos from Carnivora.

In addition to companion animals and laboratory species, about 270 carnivore species play fundamental ecological roles in different ecosystems. However, almost 40% of carnivore species are now threatened or endangered in the wild because of human activities. While protection of natural habitats is critical, it is equally important to better understand carnivore reproduction, including a solid knowledge in sperm, oocyte, and embryo biology, to maintain sustainable populations in the wild and in conservation breeding centers. Characterizing gamete and embryo biology is also needed to develop cryopreservation and assisted reproductive technologies to enhance conservation efforts. The objective of this review is to provide the most recent knowledge in the biology of sperm cells, oocytes, and early embryos across all carnivore families. Overall, most data originate from populations maintained in breeding centers or zoos. Characterizations of sperm biology and cryopreservation are far more advanced than for oocytes and embryos. Currently, sperm biology is mainly studied in Canids, Felids, Ursids, and Mustelids, with more emphasis on structural than functional properties. Importantly, fundamental studies of gamete and embryo biology in domestic dogs, cats, and ferrets have paved the way for more precise characterizations in wild counterparts as well as the development of cryopreservation and assisted reproductive technologies. A striking feature of spermatozoa across a wide range of Canids and Felids is the presence of teratospermia (>60% of abnormal sperm cells), which is related to the loss of genetic diversity in some populations. Although sperm structures differ across carnivore families, sperm biology remains difficult to compare because of the small amount of data in many species. Regarding oocyte biology and embryology, data are much scarcer than in sperm cells, with too few studies going beyond structural descriptions. More carnivore species and more individuals (especially from wild populations in addition to captive ones) must be studied to improve our understanding about comparative germplasm biology and develop adequate conservation breeding strategies including the use of cryobanking and assisted reproductive technologies.

The Knowns and Unknowns about Epididymal Extracellular Vesicles in Different Animal Species.

Sperm maturation during epididymal transit is a long and complex process. Although the roles of epididymal extracellular vesicles (EVs) on sperm quality have been extensively studied in recent years, there are still a lot of unexplored areas and too few species that are studied. The objective of this review is to focus on the contribution of epididymal EVs through the apocrine secretion of key factors, including proteins and small RNAs. Furthermore, the authors explore the alterations in the content of these vesicles related to male fertility and the effects of environmental stressors, and how these factors vary across taxa. Last, potential applications are covered, and the next steps in that field of research are highlighted.

Sperm Morphology and Male Age in Black-Throated Blue Warblers, an Ecological Model System.

Extra-pair paternity may drive selection on spermatozoa and ejaculate characteristics through sperm competition and cryptic female choice. Here, we examine sperm morphology in the black-throated blue warbler (Setophaga caerulescens), an ecological model species where extra-pair paternity is frequent and is linked with male age. We test whether sperm morphology relates to several aspects of male phenotype known or suspected to affect extra-pair paternity success. Sperm morphology did not correlate with the size of the white wing spot, a social status signal, nor with the volume of the cloacal protuberance. However, older males tended to have longer sperm cells. Although the sample size was limited, this pattern is intriguing, as longer cells may be advantageous in post-copulatory sexual selection and older males have larger testes and higher extra-pair paternity success in this species. Changes in sperm morphology with age are not observed in other birds, though they have been observed in insects and fishes. More research on sperm morphology is needed to clarify its role in extra-pair fertilizations in this well-studied species.