Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.     

The effect of catecholamines and their antagonists on the fertilization of cumulus-free mouse ova in vitro at a suboptimal spermatozoal density

A suboptimal sperm concentration was used to assess the capacity of catecholamines to stimulate the fertilization of cumulus free F₁,(C57BL × CBA) mouse ova in vitro. At a concentration of 50 μM, (L) epinephrine significantly increased the proportion of ova fertilized at 2 × l0⁵ spermatozoa/ml. However, when (D, L) propranolol at an equimolar concentration was tested for inhibition of the (L) epinephrine effect, fertilization was inhibited in both the test and control dishes. At l0μM, propanolol by itself or in the presence of 50μM (L) epinephrine significantly increased the number of ova fertilized at 2 × l0⁵ sperm/ml. Norepinephrine (50 μM) and phentolamine (50 μM), either alone or together, were also slightly stimulatory. Some data are presented to suggest that propranolol may act in a nonadrenergic manner to precipitate the acrosome reaction and that the stimulatory effect is maximised when it is added to spermatozoa at the same time as ova addition. It was suggested that propranolol may act to trigger calcium influx by a nonspecific alteration in membrane function for example in (Ca + Mg) ATPase activity. It was concluded that spermatozoa at suboptimal densities are capable of achieving fertilization and that sperm concentration dependency in fertilization in vitro may be a reflection of the proportion of spermatozoa achieving capacitation.     

Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22.4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen-thawed ICR spermatozoa was significantly improved (35.5%) by addition of glycerol (1.75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1.75% glycerol, although the fertilization rates of frozen-thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen-thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% +/- 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.     

Effect of time of ovulation on fertilization after intrabursal transfer of spermatozoa (ITS): improvement of a new method for artificial insemination in mice

The timing of AI in relation to ovulation was examined to improve intrabursal transfer of spermatozoa (ITS) in mice, a new method of AI that involves transfer of spermatozoa into a space near the infundibulum. Two microliters of fresh epididymal B6C3F1 spermatozoa (containing 2 x 10(5) spermatozoa) were inseminated 1, 7, 12, or 17 h after hCG administration. At 1.7 days after ITS, normal cleaving embryos were recovered at rates ranging from 6 to 50% (21.5 +/- 15.8%; mean +/- S.D.), 40-100% (75.2 +/- 20.2%), 33-100% (60.1 +/- 19.3%), and 6-47% (22.7 +/- 13.3%), respectively. The rate obtained by ITS 7h after hCG administration was comparable (P > 0.05) to that (90.5 +/- 6.3%) for embryos obtained after natural mating (control), but rates at all other times were significantly less than control. To examine whether in vivo fertilization rate differs when spermatozoa from various mouse strains are used, B6C3F1 females were inseminated with spermatozoa from ICR, C57BL/6N and C3H/HeN mice 7 h after hCG administration. There were strain differences (P < 0.01 for ICR and B6C3F1 versus C57BL/6N and C3H/HeN) for in vivo fertilization rates (83.9 +/- 10.3%, 75.2 +/- 20.2%, 33.6 +/- 24.5% and 25.6 +/- 16.1% for ICR, B6C3F1, C57BL/6N and C3H/HeN, respectively). Similar rates (72.9 +/- 7.3% and 27.5 +/- 46.2% for ICR and C57BL/6N, respectively) were also obtained when oocytes were inseminated with spermatozoa of the same strain. In addition, females (B6C3F1) inseminated by ITS of fresh B6C3F1 spermatozoa 7 h after hCG administration yielded normal mid-gestational fetuses with an average litter size of 7.0 +/- 4.9, which seemed much higher than the previously reported litter size of 3.2. In conclusion, the timing of AI was considered a key factor affecting in vivo fertilization efficiency.     

The relationship between sperm concentration and fertilization in vitro of rabbit ova

Different concentrations of in utero incubated rabbit sperm (1.5 × 10⁴-120 × 10⁴ /ml) were tested to determine whether there is a relationship between sperm concentration and level of fertilization achieved “in vitro” of rabbit ova. While low concentrations (1.5 × 10⁴-4.5 × 10⁴ /ml) resulted in relatively low fertilization (23–36%), those in the range of 13 × 10⁴?120 × 10⁴ /ml gave fertilization rates of 65–83%. Consistently high results were obtained with sperm counts above 40 × 10⁴ /ml. This is in agreement with the concentration of spermatozoa found in vivo in the Fallopian tubes around the time of fertilization (50 × 10⁴ /ml).     

Genetic influences on mouse sperm capacitation in vivo and in vitro

Intial in vivo studies were performed to observe the proportion of eggs fertillized at specific intervals after natural mating and ovulation in our research mouse colony. Proestrous females of the C57BL/10Wt, SJL/Wt inbred strains and the F₁ hybrid cross (B10 × SJL or reciprocals) were paired in the after-noon with males of their respective strain and examined for vaginal plugs at the midpoint of the dark period (2400 hours). Oviducts were periodically collected from mated females, and ovulation was first observed at 4, 5.2, and 3 hours after 2400 hours in the B10, SJL, and F₁ hyrid, respectively. The clutch of eggs from each ovulating female, was placed in culture, and cleavage oviduct lavage verifying female mating was placed in culture, and cleavage was used as the criterion for fertilizaition. Fifty percent of the eggs were fertilized 2.2, 5.0, and 2.5 hours after ovulation in B10, SJL, and F₁ hybrid females, respectively. Because twice the legth of time was required to fertilize a similar proportion of eggs from the SJL strain as the F₁ hybrid, these two strains were used for determining their rate of fertilization under more fully controlled conditions in vitro. Forty-nine percent of F₁ hybrid eggs were fertilized after 4 hours incubation with SJL epididymal sperm, whereas 53% fo SJL and 56% of F₁ hybrid eggs were fertilized after only 2 hours incubation with F₁ hybrid epididymal sperm. Thus, using sperm from these two mouse strains, the amount of time required to fertilize approximately 50% of the eggs within a clutch both in vivo and vitro was very similar. These observations demonstrte teh validity of using this in vitro system for fertilization studies and confirm that the temporal events in sperm capacitation and egg penetration are dependent on the genotype of the sperm. Similarities in fertilization rates at specific times after ovulation or insemination in vitro imply that the initiationof sperm capacitation in vivo occurs near the time of ovulation and several hours after mating. We tentatively suggest that follicular fluid may be required for completion of mouse sperm capacitaiton in vivo.     

Sperm motility and fertilizing ability of frozen spermatozoa of males (XY) and neomales (XX) of perch (Perca fluviatilis)

The objective of this study was to freeze sperm of sex‐reversed females (neomales) of perch and to test their fertilization ability. Sperm used was testicular (TSN), collected from females that have been inverted by means of externally administered 17‐alpha methyltestosterone. Sperm collected from intact males (SSNM) of the same origin were used as control. Prior to freezing, both TSN and SSNM were diluted into 300 mm glucose solution at the ratio of 1 : 6 and DMSO was used as cryoprotectant (10% final concentration). Crypreservation was performed in 0.5 ml straws placed into a polystyrene box, three cm above the liquid nitrogen level for 10 min and thereafter transferred fully into liquid nitrogen. Samples were thawed in 40°C water bath for 8 s and used for the fertilization experiments. Spermatozoa concentration of fresh TSN and SSNM were estimated with 45.3 × 10⁹ and 37.8 × 10⁹ spermatozoa ml^?1, respectively. Both sperm velocity and motility showed significant decreases in the TSN (134.6 μm s^?1 and 12.8%) compared to the SSNM (203.2 μm s^?1 and 94.7%) at 10 s after sperm activation. However, no differences were observed in terms of hatching rates between fresh TSN and SSNM (42.5 vs 49.3%) at fertilization densities of 12 × 10⁵ spermatozoa per egg. Frozen/thawed SSNM exhibited similar hatching rates at 12 × 10⁵ and 2.4 × 10⁵ spermatozoa per egg (37.2% vs 29.1%). Hatching rates for frozen/thawed TSN were about 7.3% with 12 × 10⁵ spermatozoa per egg and did not show any difference at 2.4 × 10⁵ spermatozoa per egg (6.6%). Stripped sperm of normal perch can be successfully frozen. Squeezing of the testes is not a good method for collection of testicular sperm resulting into low velocity, motility and hatching rate. To understand the influences of neomales on sperm quality on reproductive success further studies should be performed addressing a full assay of motility and fertility criteria when using stripped sperm from normal males and neomales. Additionally, the results indicate that many of sex reversed perch neomales are not able to release sperm and that for further studies some well spermiating neomales must to be selected.     

Relationship between semen characteristics and fertility in electroejaculated mice

Ejaculates were obtained from C57BL mice by applying two successive series of electrical stimuli which were delivered via a bipolar rectal probe. The ejaculates thus collected contained fertile spermatozoa as indicated by results from in-vitro fertilization. Once separated from the seminal plasma, ejaculated spermatozoa possessed the same in-vitro fertilization rate as epididymal sperm. Ejaculates were analysed for coagulum weight, ejaculate volume, sperm count, sperm motility, acid phosphatase content and fructose content. Significant differences were present between several of these values for fertile and infertile mice, and values were therefore empirically assigned to represent minimal amounts for 'normal' fertility (1.5 microliters ejaculate volume; 10.2 mg coagulum weight; 2.5 x 10(6) spermatozoa/ml; 2.3 x 10(3) motile spermatozoa/ejaculate). One half of the fertile animals had no deficiencies in any of the characteristics measured, whereas 97% of the infertile animals had at least one deficiency. No fertile male had more than 2 deficiencies. These data show that the characteristics of mouse semen obtained by the present method of electroejaculation are related to the fertility status of the animal.     

Le prelevement de sperme deferentiel couple a une Fecondation In Vitro dans les troubles de l’ejaculation

Surgical recovery of spermatozoa from the vas deferens is a simple and reproductible treatment for men with ejaculatory failure. After washing on a Percoll gradient spermatozoa can be used for in vitro fertilization (IVF). Also, when sperm recovery is good, surplus spermatozoa may be frozen. The indications for this treatment include retrograde ejaculation and anejaculation after classic treatments have failed and after failed vasovasostomy. During a one year period five patients were treated in this way (three with retrograde ejaculation and two with anejaculation). The five IVF attemps were performed with aspirates containing 30 to 55 × 106 spermatozoa/ml (mean=42 × 10⁶/ml), 25 to 55% motile spermatozoa (mean=43%). An average of 8.4 ovocytes were inseminated (range=8 to 21) with a fertilization rate of 67% and a success rate of 80% (one term pregnancy, two third trimester pregnancies and one second trimester pregnancy). Surgical sperm aspiration from the vas deferens in cases of ejaculatory failure is a simple, efficacious method by which sufficient mature spermatozoa for an IVF attempt (and/or cryopreservation) can be obtained     

Effects of caffeine,Ca++, capacitation time,and strain on in vitro fertilization in mice

Oocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F₁) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F₁ or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca⁺⁺ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 10⁵ spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days. B6D2F₁ spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F₁ spermatozoa capacitated in medium with 1.8 mM Ca⁺⁺ fertilized more ova (P<0.01), 83.1%, than when no Ca⁺⁺ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F₁ spermatozoa were higher, 80.1%, with a 2-hour capacitation time than with a 1-hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1-hour and 34.1% for 2-hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F₁ (26.8%) than by SJL spermatozoa (17.7%).     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures

The transportation of cryopreserved spermatozoa is an economical, efficient, and safe method for the distribution of mouse strains from one facility to another. However, spermatozoa from some strains, including C57BL/6 (B6), are very sensitive to freezing and thawing and frequently fail to fertilize eggs by conventional in vitro fertilization methods at the recipient mouse facility. Since many genetically engineered mice have the B6 genetic background, this sensitivity poses a major obstacle to studies of mouse genetics. We investigated the feasibility of transporting spermatozoa within epididymides under non-freezing conditions. First, we examined the interval that B6 and B6D2F1 (BDF1) spermatozoa retained their ability to fertilize when stored within epididymides at low temperatures (5 degrees C or 7 degrees C). Fertilization rates were >50%, irrespective of the spermatozoa used, when epididymides were stored for 3d at 7 degrees C. B6 spermatozoa, but not BDF1 sperm, had better retention of fertilizing ability at 7 degrees C versus 5 degrees C. We then transported freshly collected B6 and BDF1 epididymides from a sender colony to a recipient colony using a common package delivery service, during which the temperature was maintained at 5 degrees C or 7 degrees C for 2d. Sufficiently high fertilization rates (68.0-77.5%) were obtained for all experimental groups, except for B6 spermatozoa transported at 5 degrees C. These spermatozoa were successfully cryopreserved at the recipient facility and, yielded post-thaw fertilization rates of 27.6-66.4%. When embryos derived from the B6 spermatozoa that were transported at 7 degrees C were transferred into recipient females, 52.7% (38/72) developed to term. In conclusion, transportation of epididymides at refrigerated temperatures is a practical method for the exchange of mouse genetic resources between facilities, especially when these facilities do not specialize in sperm cryopreservation. For the B6 mouse strain, the transportation of epididymides at 7 degrees C rather than 5 degrees C, is recommended.     

Efficacy of TEPA as a “sperm label” for competitive fertilization studies in the rabbit

In this paper, the conditions necessary to use TEPA [tris (1-aziridinyl)] effectively as a label for spermatozoa in competitive fertilization are established. The fertilizing ability of rabbit spermatozoa treated with 0 and 0.8 mg TEPA/ml was compared at insemination doses of 1, 5, 20, and 40 × 10⁶ spermatozoa. Fertility was assessed by collecting ova from 64 does 48 to 52 h after insemination. TEPA blocked all but 4% of the ova from developing when 1 × 10⁶ spermatozoa were inseminated, but fertility was reduced. When 5 × 10⁶ spermatozoa were inseminated following treatment with 0, 0.6 or 1.2 mg of TEPA/ml, the fertility was 83, 74 and 50% (P<0.05), and the percentage of ova containing more than four blastomeres was 83, 11 and 5% (P<0.05), respectively. The 0.6% TEPA level was selected for a competitive fertilization trial. Equal numbers of sperm from pure Dutch-color and albino sires were combined so that either both types were untreated, only the ‘albino’ semen was treated, only the ‘Dutch’ semen was treated, or both were treated. Does were inseminated with 5 × 10⁶ sperm and allowed to kindle. The litter sizes were 5.6, 3.1, 2.7, and 0 young, and the proportion of Dutch-color progeny was 63, 97, 0 and 0%, respectively, confirming the effectiveness of TEPA as a “label”. Only one of 60 young born resulted from fertilization by a TEPA-treated spermatozoon, demonstrating that few embryos fully escape the TEPA block. Thus, the TEPA concentration and sperm numbers were established to use TEPA effectively as a label for spermatozoa in competitive fertilization studies.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.     

Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.

The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome.     

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca²⁺. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca²⁺ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca²⁺ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca²⁺ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca²⁺ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley-Liss, Inc.     

Decrease of fertilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury

In general, the fertilizing ability of cryopreserved mouse spermatozoa is less than that of fresh spermatozoa. This ability is especially low in C57BL/6, the main strain used for the production of transgenic mice. To solve this problem, the relationship between cell damage and fertilizing ability in cryopreserved mouse spermatozoa was examined in this study. Sperm motility analysis revealed no significant difference among the motilities of cryopreserved C57BL/6J, BALB/cA, and DBA/2N sperm (67.6%, 43.4%, and 60.0%, respectively) after thawing. However, the results of in vitro fertilization (IVF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) showed a strong correlation between the frequency of aberrant spermatozoa (FAS) and fertilization rates (FR; C57BL/6J: FAS, 83.7%; FR, 17.0%; BALB/cA: FAS, 67.2%; FR, 24.2%; and DBA/2N: FAS, 10.2%; FR, 93.6%), and damage to spermatozoa was localized particularly in the acrosome of the head and mitochondria.