Cengiz Yildiz

Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse

Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen-thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milk + 0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14 + PI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P < 0.05-0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P < 0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P < 0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm.

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hanks balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen-thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen-thawed-graft groups (p<0.05). Fresh grafts and frozen-thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p>0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p<0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p<0.05). The typical damage observed in the frozen-thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.

Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (Cyprinus carpio) sperm.

Addition of cholesterol-loaded cyclodextrin (CLC) to the diluents of mammalian semen increased stability and rigidity of phospholipid hydrocarbon chains of plasma membrane during sperm cryopreservation process. CLC has been tested successfully as cryoprotectant in various livestock sperm cryopreservation protocols but its efficacy for cryopreserving of fish sperm has not previously been tested. In the present study, different cholesterol loaded cyclodextrin concentrations were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with Ovopel. The extenders were prepared by using 300 mM glucose and 10% DMSO supplemented with different concentrations of CLC (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0mg per 120×10(6) spermatozoa) and without CLC (control). The pooled semen was diluted separately at a ratio of 1:3 (v/v) by using CLC extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4°C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1×10(5) spermatozoa/egg. Fresh sperm with no treatment showed the greatest sperm motility, duration of motility, viability, and fertilization results compared to the other tested cryopreserved and control groups (p<0.05). Supplementation of 1.5 mg CLC to the extender showed the best cryoprotective effect for sperm motility, duration of motility, and viability against freezing damage in comparison to extenders containing 2.5 mg, 3.0 mg CLC, and control group (p<0.05). Cryopreserved sperm containing 1.5 mg CLC provided greater result in term of fertilization success when compared to other extenders containing 0.5, 2.5, and 3.0 mg CLC or control (p<0.05). The amount of CLC effected post-thaw sperm quality and fertility as a dose-dependent manner. It is concluded that treatment of cholesterol-loaded cyclodextrin for carp sperm cryopreservation significantly improves cell cryosurvival and fertilization.

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm.

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10(5)spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p<0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p<0.05). It is concluded that the animal protein-free extender containing 10% soybean lecithin has a similar cryoprotective actions with conventional egg yolk-based extender against freezing damages and fertilization. Therefore, soybean lecithin is a suitable alternative to avian egg yolk for the cryopreservation of fish sperm.

Fresh and frozen–thawed sperm quality, nuclear DNA integrity, in vitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765mg/kg C57BL6/J or 600mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P<0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P<0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P<0.05).

Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero

PURPOSE We examined the effects of long-term hCG stimulation on germ cell maturation, and Sertoli and Leydig cell function in a xenotransplantation model of the human fetal testis. MATERIALS AND METHODS A total of 20 human fetal testes were ectopically xenografted on 20 castrated NCr male nude mice. Grafts were collected for analysis 24 weeks later. Mice were treated with saline as the control or with hCG beginning 4 weeks after the grafts were transplanted. RESULTS Of the grafts 65% survived at 24 weeks. In contrast to untreated pregrafted samples, hCG stimulated xenografts showed significantly increased density of seminiferous tubule formation with Sertoli cell migration to the basement membrane. Germ cell proliferation and differentiation from gonocytes (M2A(+)) to prespermatogonia (MAGE-4A(+)) were observed in graft samples recovered from the hCG and nonhCG treated groups at 24 weeks of treatment. Leydig cells in hCG treated grafts produced significantly more testosterone than nonhCG treated grafts. Although further studies are required to investigate the potential for further differentiation and maturation of xenografted human fetal testes, normal in utero testicular development was reproduced under long-term hCG stimulation. CONCLUSIONS This model represents a means to study long-term effects of gonadotoxins or hormonal stimulation on the maturation of human fetal testes.

Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress.

The first objective was to compare sperm quality following conventional manual sperm freezing (cryovials held 1, 2, 3, and 4 cm, respectively, above liquid nitrogen (LN(2)) for 10 min, resulting in cooling velocities of approximately -14.9, -10.1, -6.6, and -5.1 °C/min, respectively), and cooling velocities of -5, -20, -40, and -100 °C/min in a programmed automated freezer, for sperm recovered from CD-1, B6129SF1, and C57BL/6NCrlBR mice. Furthermore, using these strains, as well as 129S/SvPaslco, and DBA/2NCrlBR mice, the second objective was to determine the effects on DNA integrity of sperm exposed to hyposmotic (1 mOsm/L) and hyperosmotic (2400 mOsm/L) solutions, compared to an isosmotic control (300 mOsm/L). For freezing above LN(2) or in an automated freezer, 2 cm above LN(2) and -100 °C/min, respectively, were optimal (P < 0.05-0.01), with no significant differences between these two approaches for post-thaw progressive motility, DNA integrity, and in vitro rates of fertilization and blastocyst formation. Both manual and automated freezing techniques increased post-thaw sperm DNA fragmentation (P < 0.01); the DNA integrity of post-thaw sperm was significantly affected by cooling velocity and strain background. Relative to isosmotic controls, a hyposmotic solution was more deleterious (P < 0.05-0.01) to sperm DNA integrity than a hyperosmotic solution for CD-1, B6129SF1, C57BL/6, and DBA mice (there were strain-dependent differences). In conclusion, optimization of freezing distance and cooling velocity (manual and automated freezing, respectively) were significant factors for efficient cryopreservation and re-derivation of mice from frozen-thawed sperm. Additionally, osmotically-driven volume changes in mouse sperm increased DNA fragmentation, with susceptibility affected by background strain.