Trim66's paternal deficiency causes intrauterine overgrowth.

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66's role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

Oral D-Aspartate Treatment Improves Sperm Fertility in Both Young and Adult B6N Mice.

D-Aspartate (D-Asp) treatment improved the fertility of young male C57BL/6N mice in vivo revealing a direct role on capacitation, acrosome reaction, and fertility in vitro in young males only. We investigated whether the positive effect of D-Asp on fertility could be extended to adult males and evaluated the efficacy of a 2- or 4-week-treatment in vivo. Therefore, 20 mM sodium D-Asp was supplied in drinking water to males of different ages so that they were 9 or 16 weeks old at the end of the experiments. After sperm freezing, the in vitro fertilization (IVF) rate, the birth rate, hormone levels (luteinizing hormone (LH), epitestosterone, and testosterone), the sperm quality (morphology, abnormalities, motility, and velocity), the capacitation rate, and the acrosome reaction were investigated. Oral D-Asp treatment improves the fertilizing capability in mice regardless of the age of the animals. Importantly, a short D-Asp treatment of 2 weeks in young males elevates sperm parameters to the levels of untreated adult animals. In vivo, D-Asp treatment highly improves sperm quality but not sperm concentration. Therefore, D-Asp plays a beneficial role in mouse male fertility and may be highly relevant for cryorepositories to improve mouse sperm biobanking.

The impact of five years storage/biobanking at -80°C on mouse spermatozoa fertility, physiology, and function.

BACKGROUND: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a -80°C freezer, maintaining their ability to fertilize mouse eggs. OBJECTIVES: The main objective here was to evaluate the effects of five years at -80°C compared to liquid nitrogen storage (LN2 , control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. MATERIALS AND METHODS: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR-14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3-AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. RESULTS: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at -80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2 . DISCUSSION: Long-term mouse sperm storage at -80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. CONCLUSION: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a -80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.

Graphene Oxide Improves in vitro Fertilization in Mice With No Impact on Embryo Development and Preserves the Membrane Microdomains Architecture.

During the latest years, human infertility worsened all over the world and is nowadays reputed as a global public health issue. As a consequence, the adoption of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilization (IVF) is undergoing an impressive increase. In this context, one of the most promising strategies is the innovative adoption of extra-physiological materials for advanced sperm preparation methods. Here, by using a murine model, the addition of Graphene Oxide (GO) at a specific concentration has demonstrated to increase the spermatozoa fertilizing ability in an IVF assay, finding that 0.5 µg/ml GO addition to sperm suspensions before IVF is able to increase both the number of fertilized oocytes and embryos created with a healthy offspring given by Embryo Transplantation (ET). In addition, GO treatment has been found more effective than that carried out with methyl-ß-cyclodextrin, which represents the gold standard in promoting in vitro fertility of mice spermatozoa. Subsequent biochemical characterization of its interaction with male gametes has been additionally performed. As a result, it was found that GO exerts its positive effect by extracting cholesterol from membranes, without affecting the integrity of microdomains and thus preserving the sperm functions. In conclusion, GO improves IVF outcomes in vitro and in vivo, defining new perspectives for innovative strategies in the treatment of human infertility.

d-aspartate treatment in vitro improves mouse sperm fertility in young B6N mice.

We previously reported that the administration of d-aspartate (D-Asp) in drinking water over a 2-4-week period to 7-week-old mice resulted in higher sperm quality and increased in vitro fertilisation (IVF) rates associated with a systemic increase of luteinizing hormone and testosterone levels in the serum. The goal of this study was to investigate the effects of in vitro treatment with D-Asp on the IVF rate, embryo transfer, and sperm parameters of cryopreserved-thawed C57BL/6NTacCnrm (B6N) spermatozoa derived from young and adult mice. In this study, cryopreserved-thawed B6N spermatozoa from males aging 9, 11, 13, and 16 weeks were treated for 1 h with 4 mM D-Asp during capacitation. Thereafter, the in vitro fertilisation ability and the embryo transfer efficiency were analysed. Also, the kinetic activity of the treated spermatozoa and the acrosome reaction were measured after 1 h, 2 h, and 5 h of incubation. The capacitation rate of spermatozoa was determined after 1 h of pre-incubation. Spermatozoa from 9- and 11-week-old mice, which were treated with D-Asp, led to significantly increased IVF rates. However, spermatozoa derived from 13- and 16-week-old mice did not lead to a significant improvement in the fertilisation rate. At all ages examined, no differences were observed in the birth rate and sperm kinetic parameters. After 1 h incubation under the same conditions as the IVF was performed, the capacitation rate and the acrosome reaction were significantly higher with the D-Asp-treated spermatozoa from 9-week-old (67.5% vs. 41% and 14.5% vs. 10.5%, respectively) and 11-week-old mice (78.5% vs. 41.1% and 21.0% vs. 3.8%, respectively), corresponding to the improved IVF results. Therefore, the present results demonstrate, for the first time, a direct role of D-Asp in the capacitation process and acrosome reaction.

Effects of oral d-aspartate on sperm quality in B6N mice.

The goal of this study was to investigate the effects of oral administration of d-aspartate (D-Asp) to sexually immature male C57BL/6NTacCnrm (B6N) mice on the in vitro fertilisation (IVF) rate with cryopreserved-thawed spermatozoa and on cryopreserved sperm quality as well as on LH, epitestosterone, and testosterone levels. Males were treated at 7 weeks of age with a dose of 20 mM sodium d-aspartate in drinking water for 1.3, 2, 4 or 6 weeks so that they were 8.3, 9, 11 or 13 weeks of age, respectively, at the end of the study. The timepoints for controls were week 0 (start of experiment), 1.3, 2, 4 or 6, whereby mice received no D-Asp. At each timepoint, spermatozoa were cryopreserved for IVF and testes as well as sera were frozen for hormone level measurement. After 2 and 4 weeks of treatment, the IVF rate was significantly higher in the D-Asp group than in the controls (64% vs. 44% and 52% vs. 38%, respectively). Spermatozoa from D-Asp-treated males showed lower morphological abnormalities than their control counterparts. After 2 and especially after 4 weeks of treatment, the hormone levels in sera and testes and the total and progressive motility of the spermatozoa were higher in D-Asp-treated males. Although we did not elucidate the full mechanism leading to an improved IVF rate with spermatozoa from D-Asp-treated males lower morphological abnormalities and increased motility contribute to this observation. Importantly, D-Asp significantly improved the IVF rates as early as 2 weeks after treatment when mice were only 9 weeks old. This implies that sperm can be efficiently cryopreserved from younger males, compared to 13-weeks-old males, which are usually used for sperm cryopreservation. This is of relevance when genetic alterations cause premature death in males as well as high severity levels in older mice and aids in better resource management.

A new, simple and efficient liquid nitrogen free method to cryopreserve mouse spermatozoa at -80 °C.

The mouse is widely used for biomedical research and an increasing number of genetically altered models are currently generated, therefore centralized repositories are essentials to secure the important mouse strains that have been developed. We have previously reported that spermatozoa of wild type and mutant strains frozen using standard laboratory protocols can be transported in dry ice (-79 °C) for 7 days and safely stored in a -80 °C freezer for up to two years. The objective of this new study was to compare the effects of the freezing techniques using LN2 or -80 °C freezer on fertility of frozen-thawed mouse spermatozoa. After thawing, sperm fertility was comparable (P > 0,05) between the LN2 and the -80 °C samples for at least 1 year. Furthermore, we showed that it is possible to freeze and store mouse semen directly at -80 °C and eventually transfer it to LN2 irrespective of storage time. This study is relevant because it shows for the first time that mouse spermatozoa can be efficiently frozen and stored at -80 °C with no use of liquid nitrogen for a long period of time. A new, simple, efficient and flexible, liquid nitrogen free, method was developed for freezing and maintaining spermatozoa of wild type and mutant C57BL/6N lines. Lines on this genetic background are used in collaborative research infrastructures for systematic phenotyping, e.g. the International Mouse Phenotyping Consortium (IMPC) and therefore largely cryopreserved in repositories like EMMA/Infrafrontier. The importance of this finding will be especially useful for small laboratories with no or limited access to liquid nitrogen and for laboratories generating many mouse mutant lines by CRISPR/Cas9 who do not want to saturate the limited space of a LN2 tank, using a more accessible -80 °C freezer. This study underlines, once more, that mouse spermatozoa are very resistant and can be frozen, transported, shared and stored at -80 °C for a long time without a significant loss of viability. This new approach simplifies the freezing process and facilitates the long term storage of mouse spermatozoa at -80 °C, always allowing the transfer to LN2 for indefinite storage without noticeable detrimental effects.

Long term maintenance of frozen mouse spermatozoa at -80 °C.

Maintaining mouse stocks as frozen materials offers both ethical and economical advantages over live animal breeding if the lines are not actively used. The European Mouse Mutant Archive (EMMA) promotes the archiving and distribution of important mouse models for biomedical research through the cryopreservation of their embryos and gametes. Embryo freezing in liquid nitrogen (LN2) at -196 °C has traditionally been the method of choice for archiving mouse lines. However, sperm freezing is emerging as a more convenient alternative due to the application of innovative cryopreservation and recovery protocols. In addition, frozen spermatozoa are less sensitive to post-freezing temperature fluctuations. We have previously reported that spermatozoa frozen using standard laboratory protocols can be safely stored in a -80 °C freezer or in dry ice (-79 °C) for at least seven days. We now report the extension of this period of maintenance at -80 °C up to two 2 years both for wild type and mutant strains, indicating that once frozen, mouse spermatozoa are quite resistant and can be transported, shared and stored at -80 °C for a long time without a significant loss of viability. The importance of this finding will be especially relevant for small laboratories with no constant access to liquid nitrogen and for labs generating many mouse mutant lines by CRISPR/Cas9 who do not want to saturate the limited space of a LN2 tank, using a more accessible -80 °C freezer.

Dry ice is a reliable substrate for the distribution of frozen mouse spermatozoa: A multi-centric study.

Disseminating mouse stocks as frozen materials offers both ethical and logistical advantages over live animal shipment, minimizing the welfare issues and avoiding some of the complex custom regulations that are associated with live animal transportation. Embryo freezing in liquid nitrogen (LN2) at -196 °C has traditionally been the method of choice for archiving mouse lines. However, spermatozoa freezing is emerging as a more convenient alternative due to the application of innovative cryopreservation and recovery protocols. In addition, frozen spermatozoa are less sensitive to post-freezing temperature fluctuations. Here we demonstrated that spermatozoa frozen using standard laboratory protocols can be safely stored in dry ice (-79 °C) for at least seven days. The protocol we report here is robust and has been validated in a multi-centric study involving mouse spermatozoa samples exchanged between five European Mouse Mutant Archive (EMMA) nodes. Furthermore, following shipment on dry ice the spermatozoa can be returned to LN2 for long term storage without any noticeable detrimental effect. This protocol permits frozen spermatozoa to be shared and shipped in dry ice between biorepositories, networks and scientific institutions at low cost, using common courier companies, while avoiding the complexities, risks and hazards associated with using a traditional LN2 dry-shipper.