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The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.

Semen lyophilisation is an interesting technique that might be a cheap alternative to long-term storage in liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 and demonstrated, for the first time, the birth of healthy mouse pups from epididymal freeze-dried (mouse) spermatozoa. The authors follow the lyophilisation technique, commonly used in the pharmaceutical and food industries, namely, deep freezing, which requires direct immersion of the semen sample into liquid nitrogen before vacuum drying. In this work, we focused on the freezing phase to improve and make the technique more reliable. We compared two protocols: 1) rapid freezing, where the semen is plunged directly into liquid nitrogen (LN group), and 2) slow freezing, where the sample is frozen with a freezing rate of 1°C min−1 until −50°C (SL group). Then, both frozen samples were lyophilized. Subsequently, after an interval ranging between 1 and 3 months, dry spermatozoa from LN and SL groups were used for intracytoplasmic sperm injection (ICSI), and the embryo development was evaluated at 24h (2-cell stage) and 7 days (expanded blastocyst) post-ICSI. Moreover, acrosome integrity was evaluated with Pisum sativum agglutinin (PSA) staining on part of the semen, immediately after freezing. The LN-group semen showed the acrosome completely melted, whereas the SL group showed better integrity of the acrosome, which was comparable to that of the normal frozen (vital) spermatozoa. At 24h post-ICSI the number of cleaved embryos in the SL group was higher than in the LN group (42/100 (42%) vs. 19/75 (25.3%), SL and LN, respectively; P=0.0253). The blastocyst rate 7 days after ICSI in the SL group was higher (7/100 (7%) than that in the LN group (2/75 (2.7%); P=0.0238). Our data show that lyophilisation can be conveniently achieved in ram spermatozoa without liquid nitrogen, thus simplifying the procedure. These data support the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen.

Whereas an organism’s genotype is relatively static throughout life, the epigenome is highly dynamic and can adapt, or be altered, in response to the external environment such as diet. Intrauterine exposure to nutrient availability can alter the establishment of epigenetic marks, not only in the exposed individuals, but also in their offspring. Inheritance of such an environmentally acquired phenotype by the subsequent generation occurs through epigenetic modifications in the germline. Here, using a genome-wide approach, we evaluate how modification of the maternal diet pre- (14 days) and post- (28 days) conception can affect methylation status of the sperm of male offspring. Specifically, using a sheep model, we focused on the effect of maternal undernutrition on adult sperm methylation and its long-term consequences on sperm physiology and quality. Moreover, we investigated if supplementation of folic acid, to increase the availability of methyl donors, could prevent or ameliorate the adverse uterine environment caused by maternal undernutrition. Male lambs obtained from mothers subjected to different nutritional regimens (UND: undernutrition; FA: undernutrition and folic acid supplementation) appeared normal at birth, with a comparable body weight until Day 30 postpartum. Sperm DNA methylation, obtained by reduced representation bisulfite sequencing, differed in offspring that experienced in utero undernutrition (UND and FA) compared with the control group (CTR). In particular, the number of differentially methylated regions (DMR) was lower when UND and FA groups were compared, whereas a higher number of DMR was observed by comparison of CTR with both experimental groups. In addition, a high percentage of DMR were shared between UND and FA groups when compared with CTR, clearly indicating a influence of maternal nutrition on the offspring sperm DNA methylation rearrangement. Gene ontology (GO) analysis showed variation in functional categories related to sperm functionality such as chondroitin sulfate synthesis, potassium ion import, and others related to metabolism (biotin and glucagon). Furthermore, using computer-assisted semen analysis and flow cytometric measurement, we observed lower a sperm motility index and higher incidence of chromatin structure alterations in spermatozoa collected from UND and FA groups compared with CTR. Finally, to verify the effect of such reported abnormality on lamb fertility, we used the semen for in vitro embryo production. While we obtained good quality blastocysts from all 3 groups, a reduction in the percentage of embryo development, partially compensated in the FA group, was found using spermatozoa from UND rams. Taken together, our results confirm that a nutritional stress during early mammalian development can lead to epigenetic modification in the offspring. This damage can be partially ameliorated with folic acid supplementation; however, some alteration still persists in the germline and could be passed to the next generation, with as yet unknown consequences.

Sperm freeze-drying is a revolutionary technique that resolves many of the drawback of long-term storage under liquid nitrogen. The first significant result of this method was provided by Wakayama and Yanagimachi (1998 Nat. Biotechnol. 16, 639-641, 10.1038/nbt0798-639), demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. Besides models in the mouse and rat, which are the first small mammals born from epididymal lyophilized sperm by intracytoplasmic sperm injection (ICSI), most studies in this field have used ejaculated sperm. In this work, aiming to repeat the result of Wakayama and Yanagimachi, we tried to apply this technique to epididymal spermatozoa from a large mammal (ram). Moreover, we checked the correlation between freeze-dried spermatozoa DNA integrity and embryo development. To do this, epididymal sperm from 4 rams was lyophilized in a medium containing trehalose, glucose, KCl, HEPES, and Trolox. To evaluate DNA damage and fragmentation at rehydration, part of the sperm was processed for sperm chromatin dispersion test (SCD) and two-tailed comet assay and the rest was used for ICSI. Compared with rams 1 and 3, rams 2 and 4 had higher rate of spermatozoa with intact DNA (median: 3.3% v. 16.5%, respectively), lower rate of single strand breaks (SSB; median: 94.2% v. 81.5%, respectively) and lower rate of double-strand breaks (DSB; median: 2.5% v. 2%, respectively). Embryo development following ICSI showed that blastocyst stage was reached only from rams that had sperm with more intact DNA: ram 2 (4.8%, n = 83) and ram 4 (6.3%, n = 64). Spermatozoa from rams 1 and 3 produced no blastocysts. This can be explained by the fact that rams 2 and 4 had higher rate of spermatozoa with intact DNA than rams 1 and 3. Definitively, the implication of sperm DNA damage in embryonic development should depend on the balance between the extent of sperm DNA fragmentation, the type of fragmentation (SSB or DSB), and the oocyte’s repair capacity. Rams 2 and 4 were the only rams that produced blastocyst probably because they had considerably more sperm with normal DNA; thus, it is important to select spermatozoa of the best quality to perform a good ICSI. Fragmentation of DNA due to the lyophilization process impairs embryonic development. To conclude, oocytes injected with epididymal freeze-dried ram spermatozoa can reach the blastocyst stage. These are preliminary results; more conclusive outcomes will be given following embryo transfer experiments that are now in progress.

Pluripotency is the ability of one cell to generate every cell type of the 3 germ layers, a property typically owned by embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), with some exceptions; multilineage-differentiating stress-enduring (Muse) cells are an example. Muse cells, described as pre-existing pluripotent stem cells in mesenchymal tissues (Kuroda et al. 2010) are able to form clusters from single cells in suspension culture, express pluripotency factors and differentiate into cell types of the 3 germ layers, like ESC and iPSC. In addition, Muse cells are proposed to be the only source of cells capable to generate iPSC by current methodologies (Wakao et al. 2011). However, it is unclear whether they are normally present in adult tissue, derive from precursors stem or differentiated cells, or are induced by the in vitro conditions. In our work, we tested the hypothesis that the transition from a committed (tissue) to an uncommitted (in vitro culture) environment triggers in the cells the activation of a default gene circuitry leading to pluripotency. Adult skin fibroblasts were obtained from sheep ear biopsy (n = 3) and expanded in vitro (A) or cultured in suspension in hanging drops (B) or in nonadherent dishes (C) in MEM with 10% FBS. In a subsequent experiment, clonal expansion was attempted by culturing single suspension cells in drops of medium (D). Pluripotency was assessed analysing Oct4 and Nanog expression, using real-time PCR (mRNA) and Western blotting (protein), in cultured fibroblasts compared to whole ear biopsy (30-day-old fetus was used as positive control, CTR). Furthermore, in adherent cells (A) and in clusters obtained from suspension culture (B, C, D), Oct4 and Nanog expression was compared by immunofluorescence. We found that while in the ear biopsy not one of these pluripotency markers was expressed, in in vitro-expanded fibroblasts both mRNA and protein expression was detected; mRNA expression value (mean ± s.e.m. relative to CTR) was 0.59 ± 0.18 for Nanog and 0.2 ± 0.07 for Oct4. Moreover, fibroblasts in suspension (B, C, D) were able to form clusters [obtained from 32% (16/50) of single cells, D] similar to those normally obtained with ESC, iPSC. and Muse cells. All the clusters (B, C, D) showed a more intensive signal of Oct4 and Nanog protein compared to adherent cells by immunofluorescence. In the present work we demonstrate that adult somatic cells (skin fibroblasts) express key pluripotency factors, such as OCT4 and Nanog, in both adherent and suspension culture, after removal from the tissue (ear). We can conclude that the simple in vitro culture switches on the expression of pluripotency markers in adult somatic cells. Removal from the context of the tissue probably leads the cells to lose their tissue-specific identity and acquire a new undifferentiated one, which in an optimal condition culture could result in pluripotency. Our interpretation is that reprogramming must be an automatic, default response when differentiated cells are removed from the constraints imposed by a multicellular environment.

The differentiate state of the cell may be reversed by a process called reprogramming. To date, a totipotent status is conferred to a somatic cell by nuclear transfer (SCNT) and a condition of pluripotency is conferred by induced expression of defined factors (iPSC). While the restoration of full totipotency by SCNT is rarely achieved, pluripotency by Yamanaka's factors (Oct4, c-myc, Sox-2, Klf4) is inducible, although with low efficiency, in a large set of cell in different animal models. However, the isolation of iPSC requires complex technical skills and time-consuming protocols. In our laboratory we have observed that the simple expansion of fibroblasts in culture switches on pluripotency markers such as Oct4 and Nanog (Anzalone et al. 2015 Reprod. Fertil. Dev. IETS Abstract 344). CHIR99021 is a small molecule, targeting the Wnt/β-catenin signalling pathway, which is used for stem cell culture (Li et al. 2009). CHIR99021 acts as selective inhibitor of both isoform of GSK3 α/β regulating cellular proliferation and differentiation. In this work we tested the hypothesis that the exposure to a small reprogramming molecule (CHIR99021) induces pluripotency marker expression in primary cultures of somatic cells. Sheep and mouse primary fibroblasts cultured in low oxygen and induced to enter GO (low serum, 0.5% FBS for 5 days