Pancreatic lipase-related protein 2 is selectively expressed by peritubular myoid cells in the murine testis and sustains long-term spermatogenesis.

Spermatogenesis is a complicated process of germ cell differentiation that occurs within the seminiferous tubule in the testis. Peritubular myoid cells (PTMCs) produce major components of the basement membrane that separates and ensures the structural integrity of seminiferous tubules. These cells secrete niche factors to promote spermatogonial stem cell (SSC) maintenance and mediate androgen signals to direct spermatid development. However, the regulatory mechanisms underlying the identity and function of PTMCs have not been fully elucidated. In the present study, we showed that the expression of pancreatic lipase-related protein 2 (Pnliprp2) was restricted in PTMCs in the testis and that its genetic ablation caused age-dependent defects in spermatogenesis. The fertility of Pnliprp2 knockout animals (Pnliprp2-/-) was normal at a young age but declined sharply beginning at 9 months. Pnliprp2 deletion impaired the homeostasis of undifferentiated spermatogonia and severely disrupted the development and function of spermatids. Integrated analyses of single-cell RNA-seq and metabolomics data revealed that glyceride metabolism was changed in PTMCs from Pnliprp2-/- mice. Further analysis found that 60 metabolites were altered in the sperm of the Pnliprp2-/- animals; notably, lipid metabolism was significantly dysregulated. Collectively, these results revealed that Pnliprp2 was exclusively expressed in PTMCs in the testis and played a novel role in supporting continual spermatogenesis in mice. The outcomes of these findings highlight the function of lipid metabolism in reproduction and provide new insights into the regulation of PTMCs in mammals.

Sex-based differences in the prevalence of acute mountain sickness: a meta-analysis.

BACKGROUND: When lowlanders rapidly ascend to altitudes > 2500 m, they may develop acute mountain sickness (AMS). The individual susceptibility, ascending velocity, time spent at altitude, activity levels and altitude reached are considered risk factors for AMS. However, it is not clear whether sex is a risk factor. The results have been inconclusive. We conducted a meta-analysis to test whether there were sex-based differences in the prevalence of AMS using Lake Louise Scoring System. METHODS: Systematic searches were performed in August 2019 in EMBASE, PubMed, and Web of Science for prospective studies with AMS data for men and women. The titles and abstracts were independently checked in the primary screening step, and the selected full-text articles were independently assessed in the secondary screening step by the two authors (YPH and JLW) based on pre-defined inclusion criteria. The meta-analysis was performed using by the STATA 14.1 software program. A random-effects model was employed. RESULTS: Eighteen eligible prospective studies were included. A total of 7669 participants (2639 [34.4%] women) were tested. The results showed that there was a statistically significant higher prevalence rate of AMS in women than in men (RR = 1.24, 95%CI 1.09-1.41), regardless of age or race. Howerver, the heterogeneity was significant in the analysis (Tau2 = 0.0403, Chi2 = 50.15, df = 17; I2 = 66.1%, P = 0.000), it was main caused by different numbers of subjects among the studies (coefficient = - 2.17, P = 0.049). Besides, the results showed that there was no evidence of significant publication bias in the combined studies on the basis of Egger's test (bias coefficient = 1.48, P = 0.052) and Begg's test (P = 0.130). CONCLUSIONS: According to this study, the statistically significant finding emerging from this study was that women have a higher prevalence of AMS. However, the authors could not exclude studies where patients were on acetazolamide. Our analysis provided a direction for future studies of the relationship of sex and the risk of AMS, such as the pathological mechanism and prevention research.

Toxicity and related mechanisms of dihydroartemisinin on porcine oocyte maturation in vitro.

Dihydroartemisinin (DHA), the main active metabolite of artemisinin, has been used to treat malaria and has anticancer activities. Previous work has shown that DHA has negative impacts on embryos in rodents and primates. However, whether DHA has adverse effects on oocyte maturation is unknown. In the present study, we evaluated the toxic effects and possible mechanisms of DHA on porcine oocyte maturation. The results showed that exposure to DHA inhibited porcine oocyte polar body extrusion, and blocked cell cycle progression. Meanwhile, early embryo development after parthenogenetic activation was also impaired. DHA disturbed spindle morphology and actin assembly in porcine oocytes by reducing phosphorylation levels of MAPK. Moreover, the ROS content was increased and the mitochondrial membrane potential decreased in oocytes treated with DHA. DHA also increased the levels of intracellular and mitochondrial calcium. Furthermore, Annexin V-FITC staining showed that early apoptosis occurred in DHA-treated oocytes. The mRNA levels of apoptosis-related genes BAX and CASP3 were increased, and the anti-apoptotic gene BCL2 was decreased in oocytes exposed to DHA. Taken together, these results indicate that DHA exposure impairs porcine oocyte maturation in vitro via mechanisms involved in cytoskeleton dynamics, oxidative stress, calcium homeostasis, and apoptosis.

The Extracellular Calcium-Sensing Receptor (CASR) Regulates Gonadotropins-Induced Meiotic Maturation of Porcine Oocytes.

Gonadotropins and epidermal growth factor (EGF) play crucial roles in promoting oocyte maturation. The regulatory network downstream of these key factors is not well understood. The present study was designed to investigate the role of the calcium-sensing receptor (CASR) in porcine oocyte in vitro maturation. CASR expression was up-regulated in oocytes matured in gonadotropin-containing medium. Cortical distribution of CASR was enhanced with gonadotropins but not EGF. Supplementation of a CASR agonist (NPS R-568) in the gonadotropin (FSH and/or LH)-containing maturation medium significantly enhanced oocyte nuclear maturation. Addition of NPS2390, a CASR antagonist, compromised oocyte nuclear maturation. Furthermore, increased cortical distribution and decreased expression of CASR was observed after the NPS R-568 treatment. Oocytes treated with NPS R-568 had higher concentration of CYCLIN B1, decreased reactive oxygen species, and increased glutathione levels, indicative of advanced cytoplasmic maturation. In contrast, NPS2390 treatment compromised oocyte cytoplasmic maturation. A higher blastocyst formation rate after parthenogenetic activation was observed when oocytes were matured in the presence of the CASR agonist, NPS R-568. MAPK3/1 phosphorylation was increased during in vitro maturation and after NPS R-568 treatment, and decreased following CASR antagonist supplementation. Taken together, our data showed that the CASR is a gonadotropin-regulated factor that promotes porcine oocyte maturation in a MAPK-dependent manner.

Effect of oocyte vitrification on deoxyribonucleic acid methylation of H19, Peg3, and Snrpn differentially methylated regions in mouse blastocysts.

OBJECTIVE: To examine whether mouse oocytes vitrification could alter the deoxyribonucleic acid (DNA) methylation of differentially methylated regions (DMRs) of three imprinted genes in in vitro fertilized blastocysts. DESIGN: In vitro experiments using murine model. SETTING: State key laboratory and university research laboratory. ANIMAL(S): Kunming white mice. INTERVENTION(S): The mouse metaphase II oocytes were vitrified. After thawing, the surviving oocytes were fertilized in vitro to produce blastocysts. The blastocysts derived in vitro from fresh oocytes were used as a control. The DNA methylation patterns of the DMRs of imprinted genes in oocytes and blastocysts and the relative expression of DNMTs (Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l) in oocytes and blastocysts were detected. MAIN OUTCOME MEASURE(S): Methylation patterns of DMRs of H19, Peg3, and Snrpn analyzed by bisulfite mutagenesis and sequencing. Expression levels of messenger ribonucleic acid as measured by real-time reverse-transcriptase polymerase chain reaction. RESULT(S): After oocytes vitrification, the methylation levels at H19, Peg3, and Snrpn DMRs in blastocysts were decreased. However, there was no significant difference in the percentage of hypermethylated strands at Peg3 DMRs between the vitrified and control groups. DNMTs expression in vitrified oocytes and the expression of Dnmt3b in blastocysts derived from vitrified oocytes were significantly reduced. CONCLUSION(S): Oocytes vitrification could lead to the loss of DNA methylation of imprinted genes (H19, Peg3, and Snrpn) in mouse blastocysts, which is mainly caused by the reductions of DNMTs after vitrification of oocytes.

Decreased expression of CD9 in bovine oocytes after cryopreservation and the relationship to fertilization capacity.

This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non-vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity.

Abnormal DNA methylation in oocytes could be associated with a decrease in reproductive potential in old mice.

PURPOSE: This study was designed to evaluate DNA methylation and the expression of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b and Dnmt3L) in metaphaseII (MII) oocytes and the DNA methylation of pre-implantation embryos during mouse aging to address whether such aging-related changes are associated with decreased reproductive potential in aged mice. METHODS: Oocytes (MII) from 6 to 8 weeks old female mice are referred to as the 'young group'; oocytes from the same group that were maintained until 35-40 weeks old are referred to as the 'old group.' The oocytes were fertilized both in vitro and in vivo to obtain embryos. The DNA methylation levels in the oocytes (MII) and pre-implantation embryos were assessed using fluorescence staining. The expression levels of the Dnmt genes in the oocytes (MII) were assessed using Western blotting. RESULTS: The DNA methylation levels in the oocytes and pre-implantation embryos (in vivo and in vitro) decreased significantly during the aging of the mice. The expression levels of all of the examined Dnmt proteins in the old group were lower than young group. Both the cleavage and blastocyst rate were significantly lower in the oocytes of the older mice (69.9 % vs. 80.9 %, P < 0.05; 33.9 % vs. 56.4 %, P < 0.05). The pregnancy rate of the old mice was lower than that of the young mice (46.7 % vs. 100 %, P < 0.05). The stillbirth and fetal malformation rate was significantly higher in the old group than in the young group (17.2 % vs. 2.9 %, P < 0.05). CONCLUSIONS: The decreased expression of Dnmt1, Dnmt3a, Dnmt3b and Dnmt3L in oocytes (MII) and the change of genome-wide DNA methylation in oocytes and pre-implantation embryos due to aging may be related to lower reproductive potential in old female mice.

Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures.

Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (L(p)) and permeability (P(s)) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10(-3) M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.

Histone deacetyltransferase1 expression in mouse oocyte and their in vitro-fertilized embryo: effect of oocyte vitrification.

This study was conducted to investigate the expression of Histone Deacetyltransferase1 (HDAC1) in mouse embryos derived from the vitrified-warmed oocytes. Firstly, the mouse oocytes at metaphaseII (MII) stage were randomly allocated into three groups: A untreated (control), B exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or C vitrified by open-pulled straw (OPS) method (vitrification). After warming, they were fertilized in vitro. Fresh oocytes were used as control. Expression of HDAC1 was then examined in MII mouse oocytes and embryos by immunofluorescence with anti-HDAC1 polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Results showed that after in vitro fertilization (IVF), developmental rates to two-cell embryos (39%), 4-cell embryos (35%), morula (32%) and blastocysts (26%) in cryopreserved oocytes were all significantly lower than those of fresh oocytes (P < 0.01). In addition, HDAC1 expression in the vitrified group was significantly lower (P< 0.05) than that in the control and toxicity groups at all developmental stages except for the blastocyst. Moreover, the vitrified-warmed oocytes showed significantly lower (P < 0.05) HDAC1 expression compared with that of control and toxicity groups. In conclusion, HDAC1 was expressed both in oocytes and in their in vitro-fertilized embryos. This decreased expression of HDAC1 in mouse oocytes and the embryos due to the cryopreservation may have a negative impact on embryo development.

Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes.

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 µM Forskolin for the entire 42 h (0-42) and addition of 10 µM Forskolin between 24 and 42 h only (24-42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0-42 group than for control and 24-42 group oocytes (17, 32 and 40%, respectively; P < 0.05). It was shown in Experiment 2 that Forskolin treatment from 0-42 h and from 24-42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 µm(2) intensity of fluorescence, respectively; P < 0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0-42, 65% for 24-42 and 52% for control; P < 0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0-42 h group 0%, 24-42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0-42 h group (0-42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24-42 h group 70.60 ± 2.83; P < 0.05). The 24-42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 µM Forskolin during 24-42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h.

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos.

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Deltapsi) and microtubule distribution in mouse 2-PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2-PN embryos was significantly lower than in fresh ones (67.3 +/- 3.0% vs. 84.9 +/- 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2-PN embryos without mitochondrial rings (61.7 +/- 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 +/- 2.8%). (3) Following staining by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-cyanine iodide (JC-1), most red-colored mitochondria (high Deltapsi) were distributed peripherally around pronuclei and along cell membranes of fresh 2-PN embryos. Conversely, red-colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Deltapsi) evenly distributed throughout the cytoplasm. The proportion of fresh 2-PN embryos with obvious aggregation of high Deltapsi mitochondria (84.2 +/- 2.2%) was significantly higher than that of vitrified embryos (26.7 +/- 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 +/- 3.4%) was similar to that of vitrified embryos (74.7 +/- 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2-PN embryos, events which may affect subsequent developmental viability of such embryos.

Tetraspanin CD9 in bovine oocytes and its role in fertilization.

This study was conducted in bovine to investigate whether CD9 (a member of the tetraspanin superfamily of proteins) is present on oocytes and whether it functions in sperm-oocyte binding and fusion. First, the presence of CD9 in bovine matured oocytes was examined by immunofluorescence with the anti-CD9 monoclonal antibody (mAb) and fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and the results showed that CD9 was expressed on the plasma membrane of matured oocytes. Sperm binding and fusion with oocytes was then examined by in vitro fertilization. When the zona pellucida-free matured oocytes were fertilized, both sperm binding to ooplasma and sperm penetrating into oocytes were significantly (P<0.01) reduced in anti-CD9 antibody-treated oocytes (6.3 +/- 0.7 per oocyte and 41.6%, respectively) compared with untreated control oocytes (19.0 +/- 0.7 per oocyte and 81.3%, respectively), indicating that the anti-CD9 mAb potentially inhibits sperm-oocyte binding and fusion. These results demonstrated that the CD9 present on bovine matured oocytes is involved in sperm-oocyte interaction during fertilization.

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes.

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P<0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol+vitrification, 69.98%) and Taxol pretreatment significantly (P<0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 microm). The number of lipid droplets (5-10 microm in diameter) in vitrified oocytes pretreated with Taxol was higher (P<0.05) than that in the oocytes without Taxol pretreatment (81.87+/-13.63 vs. 64.27+/-13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group. In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages.

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

Production of normal offspring from partially zona-incised vitrified mouse oocytes fertilized with cryopreserved spermatozoa using an optimized protocol.

The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group 2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized with frozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0 to 60 percent) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2 percent) and blastocysts formation rates (63.6 percent) were obtained when the egg yolk concentration was adjusted to 30 percent. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40 degree C before plunging into liquid nitrogen. After thawing, the highest cleavage rate (87.4 percent) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2 percent, 65.4 percent) and Group 3 (72.5 percent, 45.0 percent) were higher (P less then 0.05) than those in Group 2 (22.2 percent and 13.9 percent), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1 percent and 22.7 percent of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.

Piezo-assisted in vitro fertilization of mouse oocytes with spermatozoa retrieved from epididymides stored at 4 degree C.

This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa.

Conventional freezing, straw, and open-pulled straw vitrification of mouse two pronuclear (2-PN) stage embryos.

Little is known on the cryopreservation of mouse pronuclear (PN) stage embryos. In the present experiment the mouse 2-PN stage embryos were cryopreserved by conventional freezing, straw, or open-pulled straw (OPS) vitrificaiton methods. The conventional freezing solution was 1.5 mol/L ethylene glycol (EG), and vitrification solutions were EFS30 (30% EG, Ficoll, and sucrose), EFS40 (40% EG, Ficoll, and sucrose), EDFS30 (15% EG, 15%dimethyl sulfoxide [DMSO], Ficoll, and sucrose), or EDFS40 (20% EG, 20%DMSO, Ficoll, and sucrose). The blastocyst rate of 2-PN stage embryos cryopreserved by conventional method (30.4%) was lower than those vitrified by straw method with EDFS (56.9% to 69.1%), by OPS method (66.0% to 85.7%), and that of control (80.8%) (P < 0.05). With a given vitrificaiton solution EFS30, EFS40, EDFS30, or EDFS40, the blastocyst rate of embryos vitrified by the OPS method (66.7%, 66.0%, 85.7%, or 76.9%) was higher than that of those vitrified by the straw method (46.8%, 43.8%, 69.1%, or 56.9%) (P < 0.05). When mouse 2-PN-stage embryos were vitrified with EDFS30 by straw or OPS method, the highest blastocyst rate was achieved (69.1% or 85.7%) and was similar to that of the control, respectively. The embryos transfer results revealed that the full-term development of blastocysts derived from 2-PN stage embryos vitrified by OPS method with EDFS30 (19.9%) was similar to that of the control (23.5%), and higher than that of those cryopreserved by conventional freezing (9.3%) (P < 0.05). The present research demonstrates that the OPS method, especially with EDFS30, is more effective in cryopreserving mouse 2-PN embryos.

Effect of in-straw thawing on in vitro- and in vivo-development of vitrified mouse morulae.

For the purpose of ascertaining parameters to embryo transfer on some domestic animals, mouse morulae were used as a model to investigate the effect of in-straw thawing on in vitro and in vivo-development of vitrified embryos. Embryos were vitrified in 0.25 ml straws preloaded with dilution solution (0.5 M Sucrose) and thawed in the straw by mixing the vitrification solution (Ethylene glycol + Ficoll 70 + Sucrose) and the dilution solution at 25 degrees C. The embryos were randomly divided into six groups and expelled from the straws after they had been suspended in the in-straw mixture for 3 min, 5 min, 8 min, 12 min, 16 min, and 20 min, respectively, and then they were collected under a microscope for in vitro culture or direct transfer. The in vitro developmental rates of the embryos were 92.3% to 98.4% and hatching rates were 64.1% to 75.6% for the groups of 3 min to 16 min, showing no significant differences with those of nonfrozen controls (100%, 76.2%; P > 0.05). While embryos were suspended in the straw for 20 min, the developmental rate (86.6%) and hatching rate (52.4%) were significant lower than those of the control (100%, 76.2%; P < 0.01). When the 168 frozen-thawed embryos (in-straw thawing for 5 min) and 168 fresh embryos were transferred, respectively, the proportion of live fetuses in the pregnant recipients between them (58.7% vs. 54.5%) showed no significant difference (P > 0.05). The data indicate that vitrification with EFS30 and suspension in the in-straw mixture for 3 min to 16 min, when thawing, did not affect the in vitro developmental rate and hatching rate. Moreover, the in vivo developmental rate between vitrified embryos and fresh embryos did not differ significantly. It can be concluded that this method is fit for nonsurgical embryo transfer in some domestic animals with a suggestion that the operation of embryo transfer should be accomplished within 16 min.

Open-pulled straw (OPS) vitrification of mouse hatched blastocysts.

This study was first employed to investigate the developmental potential of mouse hatched blastocyts (HBs) vitrified by a two-step open-pulled straw (OPS) method. HBs were obtained by culture of morulae in vitro. First, the embryos were placed in four cryprotectant solutions - that is, 10% ethylene glycol (EG), 10%E + 10%D (10% EG and 10% dimethyl sulphoxide (DMSO) in mPBS), EFS30 (30% EG, Ficoll, and sucrose) and EDFS30 (15% EG, 15% DMSO, Ficoll, and sucrose)--at 25 degrees C for 0.5 to 10 min, respectively, to determine their optimal survival after rapid dilution in 0.5 M sucrose. Secondly, based on the above best survival, the embryos were plunged into liquid nitrogen after first pretreatment in 10%E for 0.5 min and then 0.5 min equilibration in EFS30 (Group 1), or 10%E + 10%D and EDFS30 for 0.5 min, respectively (Group 2). When warming, three methods were used to dilute the cryoprotectants from the vitrified embryos. The embryos were assessed by the re-expansion of the blastocoel or development to term. The result showed that all the vitrified-warmed HBs got high in vitro survival rates (83.7% to 98.9%). The highest in vitro survival rates (87.8% in Group 1, 98.9% in Group 2) were obtained when the vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min (method C). When the vitrified embryos diluted with method C were transferred, their survival rate in vivo (35.5% to 42.2% of the total) were similar to (P > 0.05) that of control (45.7%). These results demonstrate OPS method was highly efficient for the cryopreservation of mouse HBs.

Stepwise in-straw dilution and direct transfer using open pulled straws (OPS) in the mouse: a potential model for field manipulation of vitrified embryos.

In the present study, mouse blastocysts were employed to investigate the feasibility and efficiency of stepwise in-straw dilution and direct transfer using the open pulled straw (OPS) method. In experiment I, the effects of various vitrification solutions (VS) on embryo survival were examined. After thawing, the expanded blastocyst rates (97.59 and 95.05%) and hatching rates (80.48 and 78.95%) achieved in the EDFS30 [15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll, and sucrose] and EFS40 [40% EG, Ficoll, and sucrose] groups were no different from those (96.15% and 83.33%) of the control group. However, the rates in the EFS30 [30% EG, Ficoll, and sucrose] (87.80 and 55.43%) and EDFS40 [20% EG, 20% DMSO, Ficoll, and sucrose] (95.69 and 70.97%) groups were significantly lower than those (96.15 and 83.33%) of the control group (P<0.05). In the experiment II, the effects of the volume of VS in the OPS on the survival of embryos after in-straw thawing were investigated. When the length of the VS in the column was less than 1 cm, the in vitro viability of embryos thawed by stepwise in-straw dilution was no different among the experimental and control groups. The embryos could be successfully thawed by immersing the OPS in 0.5 M sucrose for 3 min and then 0.25 M sucrose for 2 min. In experiment III, the effect of immersion time of the OPS in diluent (PBS) on the viability of vitrified embryos was investigated. After in-straw thawing, OPSs were immersed immediately in 1 ml PBS for 0 to 30 min. When the immersion time of the OPSs in PBS was less than 12 min, in vitro development of the in-straw thawed embryos was no different from that of the controls. In experiment IV, in-straw thawed blastocysts were directly transferred to pseudopregnant mice to examine their in vivo developmental viability. The pregnancy (91.67%) and birth rates (42.42%) of embryos in-straw thawed and directly transferred were no different from those of the unvitrified controls (90.90 and 40%) and embryos thawed by the conventional method (84.61 and 46.94%). These results demonstrate that mouse embryos vitrified with OPS could be successfully thawed by stepwise in-straw dilution and transferred directly to a recipient and that this method might be a model for field manipulation of vitrified embryos in farm animals.