Cryopreservation of grey wolf (Canis lupus) testicular tissue.

Development of genomic preservation technologies for canids, especially for seasonally breeding species like the grey wolf (Canis lupus), is needed in advance of growing species conservation concerns. Here, we evaluated the efficacy of two cryopreservation protocols - needle immersion vitrification (NIV) and slow freezing (SF) on grey wolf (n = 7) testicular tissue morphology. NIV samples were equilibrated in a 7.5% v/v dimethyl sulfoxide (DMSO or Me2SO) + 7.5% ethylene glycol (EG) solution in minimum essential medium with 20% FBS for 10 min at 4 °C, then exposed to 15% DMSO + 15% EG + 0.5 M sucrose for 10 min at 4 °C before plunging into liquid nitrogen. For slow freezing, we assessed two cryoprotectant (CPA) strategies, DMSO, 15% v/v alone (SF-D) or 7.5% EG + 7.5% DMSO (SF-ED). Following thawing, there were no significant differences in seminiferous tubule area among treatment groups, although all cryopreserved tissues displayed reduced tubule size compared with fresh controls and increased apoptosis, the latter reaching significance for SF-D treated tissues. Slow freezing improved maintenance of testis architecture, with minimal detachment of seminiferous tubule basement membranes post-thaw. Spermatogonia densities were reduced in NIV tissues compared with fresh, with no differences in spermatocyte, spermatid, or Sertoli cell counts, or germ cell marker DDX4+ cell densities among groups. In sum, we conclude that slow freezing better maintained morphology of cryopreserved testicular tissues compared with needle vitrification with 15% each DMSO and EG and 0.5 M sucrose, and that DMSO + EG combination SF supports cell viability. This represents a first step in the development of male gonadal tissue preservation strategies for the grey wolf.

Spindle configuration and developmental competence of in vitro-matured bovine oocytes exposed to NaCl or sucrose prior to Cryotop vitrification.

In the present study we examined whether exposure to high concentrations of NaCl or sucrose before vitrification improves the cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, oocytes were exposed to different concentrations of NaCl (375-1517 mOsm) or sucrose (375-812 mOsm) for 1h. On the basis of the results of this experiment, in Experiment 2 oocytes were exposed to 0.25% NaCl (375 mOsmol) or 2.77% sucrose (375 mOsmol) solution, vitrified and warmed. Microtubule and chromosome configurations were examined by immunocytochemistry. In Experiment 3, in vitro embryo development was assessed after vitrification of oocytes with or without 2.77% sucrose (375 mOsmol) pretreatment. There was a similar percentage of oocytes showing normal spindle configurations in the sucrose-pretreated and control groups. Higher rates of abnormal spindles were found in groups treated with NaCl or sucrose solutions with >375 mOsmol. After vitrification and warming, a significantly higher percentage of oocytes with normal chromosome configurations was recorded for oocytes exposed to 375 mOsmol sucrose solution before vitrification compared with the control vitrified oocytes. However, these percentages were significantly lower than those recorded in untreated controls. Cleavage and blastocyst rates were higher in non-vitrified than vitrified oocytes. In conclusion, pretreatment with 375 mOsmol NaCl or sucrose solution had no adverse effects on the spindle status of vitrified-warmed cow oocytes. However, sucrose pretreatment offered no benefits for embryo development.

An oviduct-on-a-chip provides an enhanced in vitro environment for zygote genome reprogramming.

Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development.

Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production.

The oviduct was long considered a largely passive conduit for gametes and embryos. However, an increasing number of studies into oviduct physiology have demonstrated that it specifically and significantly influences gamete interaction, fertilization and early embryo development. While oviduct epithelial cell (OEC) function has been examined during maintenance in conventional tissue culture dishes, cells seeded into these two-dimensional (2-D) conditions suffer a rapid loss of differentiated OEC characteristics, such as ciliation and secretory activity. Recently, three-dimensional (3-D) cell culture systems have been developed that make use of cell inserts to create basolateral and apical medium compartments with a confluent epithelial cell layer at the interface. Using such 3-D culture systems, OECs can be triggered to redevelop typical differentiated cell properties and levels of tissue organization can be developed that are not possible in a 2-D culture. 3-D culture systems can be further refined using new micro-engineering techniques (including microfluidics and 3-D printing) which can be used to produce 'organs-on-chips', i.e. live 3-D cultures that bio-mimic the oviduct. In this review, concepts for designing bio-mimic 3-D oviduct cultures are presented. The increased possibilities and concomitant challenges when trying to more closely investigate oviduct physiology, gamete activation, fertilization and embryo production are discussed.

Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.