Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

Parental ageing and Assisted Reproduction Technologies: analysis of risk of chronic diseases in the progeny / Pózne rodzicielstwo i rozród wspomagany: analiza ryzyka wystapienia chorób przewleklych u potomstwa.

Conception of a child at advanced parental age (> 35 years) has been steadily increasing in recent decades, especially in developed countries. Socio-economic factors, effective contraceptives, and the availability of Assisted Reproduction Technologies (ART) have a direct impact on postponing the decision to have a baby. ART enables reproductive success for people diagnosed as infertile or with reduced possibilities of becoming pregnant due to concomitant pathologies. Epidemiological studies indicate that both advanced parental age and ART are associated with pathologies of pregnancy, such as gestational diabetes, risk of pre-eclampsia, miscarriage, placental abruption, preterm labor, stillbirth, neurodevelopmental disorders and chronic disease of the offspring. In our work, we will focus on the available information on metabolic changes that increase the risk of developing cardiovascular diseases in the offspring of parents at an advanced age and conceived through ART. Finally, we will address the sources of the observed disturbances at the gamete and embryo level, related to oxygen stress, epigenetic modifications and DNA damage, considering possible rescue actions.

The effects of assisted reproduction technologies on metabolic health and disease†.

The increasing prevalence of metabolic diseases places a substantial burden on human health throughout the world. It is believed that predisposition to metabolic disease starts early in life, a period of great susceptibility to epigenetic reprogramming due to environmental insults. Assisted reproductive technologies (ART), i.e., treatments for infertility, may affect embryo development, resulting in multiple adverse health outcomes in postnatal life. The most frequently observed alteration in ART pregnancies is impaired placental nutrient transfer. Moreover, consequent intrauterine growth restriction and low birth weight followed by catch-up growth can all predict future obesity, insulin resistance, and chronic metabolic diseases. In this review, we have focused on evidence of adverse metabolic alterations associated with ART, which can contribute to the development of chronic adult-onset diseases, such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Due to high phenotypic plasticity, ART pregnancies can produce both offspring with adverse health outcomes, as well as healthy individuals. We further discuss the sex-specific and age-dependent metabolic alterations reflected in ART offspring, and how the degree of interference of a given ART procedure (from mild to more severe manipulation of the egg) affects the occurrence and degree of offspring alterations. Over the last few years, studies have reported signs of cardiometabolic alterations in ART offspring that are detectable at a young age but that do not appear to constitute a high risk of disease and morbidity per se. These abnormal phenotypes could be early indicators of the development of chronic diseases, including metabolic syndrome, in adulthood. The early detection of metabolic alterations could contribute to preventing the onset of disease in adulthood. Such early interventions may counteract the risk factors and improve the long-term health of the individual.

Embryo biopsy and development: the known and the unknown.

Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting 'healthy' embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select 'healthy' embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.

Synergies between assisted reproduction technologies and functional genomics.

This review, is a synopsis of advanced reproductive technologies in farm animals, including the discussion of their limiting factors as revealed by the study of offspring derived from embryos produced in vitro and through cloning. These studies show that the problems of epigenetic mis-programming, which were reported in the initial stages of assisted reproduction, still persist. The importance of whole-genome analyses, including the methylome and transcriptome, in improving embryo biotechnologies in farm animals, are discussed. Genome editing approaches for the improvement of economically-relevant traits in farm animals are also described. Efficient farm animal embryo biotechnologies, including cloning and the most recent technologies such as genome editing, will effectively complement the latest strategies to accelerate genetic improvement of farm animals.

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development.

PURPOSE: This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI). METHODS: Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oocytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS). RESULTS: No differences were observed in the zygote/cleavage/blastocyst rate between chemically activated and non-activated oocytes (50 vs. 45 %, 11.6 vs. 10.1 %; 1.8 vs. 1.1 %, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45 %, p < 0.01; 15.5 vs. 1.1 %, p < 0.0001, respectively). Moreover, the percentage of PPS-derived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2 %). The ICSI blastocysts' development was also improved with PPS pre-treated with ionomycin (15.6 %), but was completely impeded with PPS pre-treated with Triton X-100 (0 %). CONCLUSION: Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechanically damaged substantially improves embryonic development until the blastocyst stage.

Genetic diversity of Italian goat breeds assessed with a medium-density SNP chip.

BACKGROUND: Among the European countries, Italy counts the largest number of local goat breeds. Thanks to the recent availability of a medium-density SNP (single nucleotide polymorphism) chip for goat, the genetic diversity of Italian goat populations was characterized by genotyping samples from 14 Italian goat breeds that originate from different geographical areas with more than 50 000 SNPs evenly distributed on the genome. RESULTS: Analysis of the genotyping data revealed high levels of genetic polymorphism and an underlying North-south geographic pattern of genetic diversity that was highlighted by both the first dimension of the multi-dimensional scaling plot and the Neighbour network reconstruction. We observed a moderate and weak population structure in Northern and Central-Southern breeds, respectively, with pairwise FST values between breeds ranging from 0.013 to 0.164 and 7.49 % of the total variance assigned to the between-breed level. Only 2.11 % of the variance explained the clustering of breeds into geographical groups (Northern, Central and Southern Italy and Islands). CONCLUSIONS: Our results indicate that the present-day genetic diversity of Italian goat populations was shaped by the combined effects of drift, presence or lack of gene flow and, to some extent, by the consequences of traditional management systems and recent demographic history. Our findings may constitute the starting point for the development of marker-assisted approaches, to better address future breeding and management policies in a species that is particularly relevant for the medium- and long-term sustainability of marginal regions.

Impaired placental vasculogenesis compromises the growth of sheep embryos developed in vitro.

To evaluate how assisted reproductive technologies (ART) affect vasculogenesis of the developing conceptus, we analyzed placental and fetal development of in vitro-produced (IVP) sheep embryos. Pregnancies produced by ART carry increased risk of low birth weight, though what causes this risk remains largely unknown. We recently reported that developmental arrest of sheep conceptuses obtained by ART is most pronounced when the cardiovascular system develops (Days 20-30 of development). A total of 86 IVP blastocysts (2-4 per ewe) were surgically transferred to 30 recipient sheep 6 days after estrus; 20 sheep were naturally mated (control). Conceptuses were recovered from sheep at Days 20, 22, 26, and 30 of gestation and morphologically evaluated. Then, the conceptuses and part of their placentae (chorion-allantois) were fixed for histological and immunohistochemical analysis and snap-frozen in liquid nitrogen for subsequent mRNA expression analysis. Results demonstrate that the cardiovascular systems of sheep IVP conceptuses were severely underdeveloped. Pericardial and placental hemorrhages were noted in a majority (5/7) of the dead embryos. In the surviving IVP embryos, the expression of angiogenetic factors was reduced at Day 20. The placental vessels were underdeveloped on Days 20 and 22 (P < 0.05), though placental vasculogenesis was successfully completed on subsequent days. However, low vessel number persisted at Days 26 and 30 (4.6 vs. 5.9 and 6.64 vs. 8.70 per field, respectively; P < 0.05) together with reduced vessel diameter at Day 26 (46.89 vs. 89.92 µm; P < 0.05). In vitro production of sheep embryos induced severely impaired vasculogenesis early in gestation. This may lead to developmental programing problems, such as intrauterine growth restriction of the fetus, resulting in long-term health consequences for the offspring, such as cardiovascular diseases.

Cloning the mammoth: a complicated task or just a dream?

Recently there has been growing interest in applying the most advanced embryological tools, particularly cloning, to bring extinct species back to life, with a particular focus on the woolly mammoth (Mammuthus primigenius). Mammoth's bodies found in the permafrost are relatively well preserved, with identifiable nuclei in their tissues. The purpose of this chapter is to review the literature published on the topic, and to present the strategies potentially suitable for a mammoth cloning project, with a frank assessment of their feasibility and the ethical issues involved.

Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow-derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34- and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre-clinical sheep model to test the efficiency and safety of cell replacement therapy.

Post-implantation mortality of in vitro produced embryos is associated with DNA methyltransferase 1 dysfunction in sheep placenta.

STUDY QUESTION: Is DNA methyltransferase 1 (DNMT1) dysfunction involved in epigenetic deregulation of placentae from embryos obtained by assisted reproduction technologies (ARTs)? SUMMARY ANSWER: DNMT1 expression in growing placentae of in vitro produced (IVP) embryos is compromised and associated with pregnancy loss. WHAT IS KNOWN ALREADY: DNMT1 maintains the methylation profile of genes during cell division. The methylation status of genes involved in placenta development is altered in embryos obtained in vitro. Disturbances in the epigenetic regulation of gene expression during placentogenesis could be involved in the frequent developmental arrest and loss of IVP embryos. STUDY DESIGN, SIZE, DURATION: Forty sheep were naturally mated (Group 1, CTR). IVP blastocysts (2-4 per ewe) were surgically transferred to the remaining 46 recipient sheep 6 days after oestrus (Group 2). Twenty-one recipients from Group 1 and 27 recipients from Group 2 were allowed to deliver in order to compare embryo survival in both groups at term (150 days). From the remaining recipients (n = 38), fetuses and placentae of both groups were recovered by paramedian laparotomy at Days 20, 22, 24, 26 and 28 of gestation. MATERIALS, SETTING, METHODS: Immediately after collection, early placental tissues (chorion-allantois) were snap frozen in liquid nitrogen and DNMT1 expression and activity was evaluated. mRNA levels (for DNMT1, HDAC2, PCNA, DMAP1, MEST, IGF2, CDKN1C, H19) and the methylation status of H19 were also analyzed. Furthermore, embryo size and survival rate were measured. MAIN RESULTS AND THE ROLE OF CHANCE: Our study shows that DNMT1 expression was reduced in early placentae from sheep IVP embryos. This reduction was associated with growth arrest and subsequent death of the sheep embryos. Conversely, normal levels of DNMT1 and its cofactors were observed in placentae from IVP embryos that survived this developmental bottleneck. Although DNA methylation machinery was severely compromised in IVP placentae only up to Day 24, the low DNMT1 enzymatic activity that persisted after this stage in IVP placentae was not lethal for the developing embryos. LIMITATIONS, REASONS FOR CAUTION: The studied genes represent only a small fraction of genes regulating DNA methylation. Further studies are needed to evaluate changes in the expression and methylation status of other genes that may lead to developmental arrest of IVP embryos. As this is the only study evaluating the functionality of DNMT1 machinery in placentae from ART embryos, studies on other species are needed to confirm if our observation may be applicable to all mammalian embryos produced in vitro. WIDER IMPLICATIONS OF THE FINDINGS: The knowledge about compromised activity of DNMT1 in placentae obtained from IVP embryos should stimulate detailed studies on the metabolic requirements of oocytes and embryos in order to adequately enrich the culture media.

Embryonic diapause in humans: time to consider?

BACKGROUND: When a competent blastocyst stage embryo finds itself in an unreceptive uterus, it delays development. In around one hundred species representing various orders, this delay is known to be reversible, but this phenomenon - termed embryonic diapause (ED) - is not considered a general characteristic of all mammals. PRESENTATION OF THE HYPOTHESIS: Recently, however, we demonstrated that a non-diapausing species, the sheep, is capable of ED, suggesting the hypothesis that this is in fact an ancestral trait common to all mammals, including humans. TESTING THE HYPOTHESIS: In spite of the obvious difficulties in testing this idea, we propose a combination of indirect observations on human fertility patients, and direct study of the embryos of non-human primates. IMPLICATIONS OF THE HYPOTHESIS: Support for our hypothesis would require revision of obstetric interventions routinely performed when a human pregnancy extends beyond the due date.

The ups and downs of somatic cell nucleus transfer (SCNT) in humans.

Achieving successful somatic cell nuclear transfer (SCNT) in the human and subhuman primate relative to other mammals has been questioned for a variety of technical and logistical issues. Here we summarize the gradual evolution of SCNT technology from the perspective of oocyte quality and cell cycle status that has recently led to the demonstration of feasibility in the human for deriving chromosomally normal stem cells lines. With these advances in hand, prospects for therapeutic cloning must be entertained in a conscientious, rigorous, and timely fashion before broad spectrum clinical applications are undertaken.

Nile Red and BODIPY Staining of Lipid Droplets in Mouse Oocytes and Embryos.

Lipid droplets (LDs) are intracellular structures composed of hydrophobic lipids. Their amount in oocytes and embryos varies among the mammalian species and even among different strains of the same species. Here we describe a method to stain LDs, which can be applied to previously fixed mouse oocytes and embryos. This method is based on fluorescent dyes, Nile red and BODIPY, which allow visualization and quantification of LDs using conventional and confocal fluorescence microscopy.

A short exposure to polychlorinated biphenyls deregulates cellular autophagy in mammalian blastocyst in vitro.

BACKGROUND: Polychlorinated biphenyls (PCBs) are common environmental contaminants that represent an important risk factor of reproductive disorders in chronically exposed human populations. However, it is not known whether a short accidental exposure of embryos to PCBs before implantation might influence their further development and whether the effect might be reversible. METHODS AND RESULTS: To this aim, in vitro-matured sheep blastocysts were incubated with 2 or 4 µg/ml Aroclor 1254 (A1254), a mixture of 60 PCB congeners for 48 h after which blastocyst proliferation and ability for outgrowth in vitro were assessed. Blastocysts exposed to A1254 showed: (i) reduced proliferation and cell number (particularly in the inner cell mass compartment); (ii) accumulation of vacuoles and lipid droplets, diffused mitochondrial damage and up-regulation of autophagy markers (ATG6 and LC3), all signs indicative of deregulated autophagy, and (iii) massive cell death. Although exposed embryos resumed growth following A1254 removal, their subsequent development remained severely perturbed. CONCLUSIONS: These findings indicate that short exposure of blastocysts to PCBs leads to its damage characterized by deregulated autophagy and subsequent cell death.

DNA damage in preimplantation embryos and gametes: specification, clinical relevance and repair strategies.

BACKGROUND: DNA damage is a hazard that affects all cells of the body. DNA-damage repair (DDR) mechanisms are in place to repair damage and restore cellular function, as are other damage-induced processes such as apoptosis, autophagy and senescence. The resilience of germ cells and embryos in response to DNA damage is less well studied compared with other cell types. Given that recent studies have described links between embryonic handling techniques and an increased likelihood of disease in post-natal life, an update is needed to summarize the sources of DNA damage in embryos and their capacity to repair it. In addition, numerous recent publications have detailed novel techniques for detecting and repairing DNA damage in embryos. This information is of interest to medical or scientific personnel who wish to obtain undamaged embryos for use in offspring generation by ART. OBJECTIVE AND RATIONALE: This review aims to thoroughly discuss sources of DNA damage in male and female gametes and preimplantation embryos. Special consideration is given to current knowledge and limits in DNA damage detection and screening strategies. Finally, obstacles and future perspectives in clinical diagnosis and treatment (repair) of DNA damaged embryos are discussed. SEARCH METHODS: Using PubMed and Google Scholar until May 2021, a comprehensive search for peer-reviewed original English-language articles was carried out using keywords relevant to the topic with no limits placed on time. Keywords included 'DNA damage repair', 'gametes', 'sperm', 'oocyte', 'zygote', 'blastocyst' and 'embryo'. References from retrieved articles were also used to obtain additional articles. Literature on the sources and consequences of DNA damage on germ cells and embryos was also searched. Additional papers cited by primary references were included. Results from our own studies were included where relevant. OUTCOMES: DNA damage in gametes and embryos can differ greatly based on the source and severity. This damage affects the development of the embryo and can lead to long-term health effects on offspring. DDR mechanisms can repair damage to a certain extent, but the factors that play a role in this process are numerous and altogether not well characterized. In this review, we describe the multifactorial origin of DNA damage in male and female gametes and in the embryo, and suggest screening strategies for the selection of healthy gametes and embryos. Furthermore, possible therapeutic solutions to decrease the frequency of DNA damaged gametes and embryos and eventually to repair DNA and increase mitochondrial quality in embryos before their implantation is discussed. WIDER IMPLICATIONS: Understanding DNA damage in gametes and embryos is essential for the improvement of techniques that could enhance embryo implantation and pregnancy success. While our knowledge about DNA damage factors and regulatory mechanisms in cells has advanced greatly, the number of feasible practical techniques to avoid or repair damaged embryos remains scarce. Our intention is therefore to focus on strategies to obtain embryos with as little DNA damage as possible, which will impact reproductive biology research with particular significance for reproductive clinicians and embryologists.

Ribosomal DNA methylation in human and mouse oocytes increases with age.

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages.

Species-Specific Paternal Age Effects and Sperm Methylation Levels of Developmentally Important Genes.

A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here.

Sheep (Ovis aries) Macrophage Migration Inhibitory Factor: molecular cloning, characterization, tissue distribution and expression in the ewe reproductive tract and in the placenta.

Macrophage Migration Inhibitory Factor (MIF) is a pivotal regulator of innate and acquired immunity affecting the response and behavior of macrophages and lymphocytes. However, a number of studies indicated wider physiological functions for this cytokine to include key-roles in reproductive biology. The present study was designed to clone the coding sequence of sheep MIF, to examine the characteristics of the protein in vitro, and to evaluate its expression in sheep tissues and in the ewe reproductive tract in vivo. Ovine MIF cDNA consisted of 348 nucleotides encoding a 115 amino acids protein with an estimated molecular mass of 12,343 Da and an isoelectric point of 7.68. Sheep MIF shared high amino acid identity with the other mammalian MIF family members and showed parallel functions to human MIF, displaying enzymatic oxoreductase activity and inducing monocyte transmigration. Expression studies detected a MIF transcript in all the sheep tissues examined. Among reproductive tissues, MIF mRNA and protein were detected in the ovary, oviduct, uterus and placenta. These results indicate that sheep MIF shares crucial features with other MIF family members and delineate its potential involvement in several aspects of ovine physiology.

Genome of non-living cells: trash or recycle?

Reproductive technologies have been often used as a tool in research not strictly connected with developmental biology. In this study, we retrace the experimental routes that have led to the adoption of two reproductive technologies, ICSI and somatic cell nuclear transfer (SCNT), as biological assays to probe the 'functionality' of the genome from dead cells. The structural peculiarities of the spermatozoa nucleus, namely its lower water content and its compact chromatin structure, have made it the preferred cell for these experiments. The studies, primarily focused on mice, have demonstrated an unexpected stability of the spermatozoa nuclei, which retained the capacity to form pronuclei once injected into the oocytes even after severe denaturing agents like acid treatment and high-temperature exposure. These findings inspired further research culminating in the production of mice after ICSI of lyophilized spermatozoa. The demonstrated non-equivalence between cell vitality and nuclear vitality in spermatozoa prompted analogous studies on somatic cells. Somatic cells were treated with the same physical stress applied to spermatozoa and were injected into enucleated sheep oocytes. Despite the presumptive fragile nuclear structure, nuclei from non-viable cells (heat treated) directed early and post-implantation embryonic development on nuclear transfer, resulting in normal offspring. Recently, lyophilized somatic cells used for nuclear transfer have developed into normal embryos. In summary, ICSI and SCNT have been useful tools to prove that alternative strategies for storing banks of non-viable cells are realistic. Finally, the potential application of freeze-dried spermatozoa and cells is also discussed.