An engineered enzyme embedded into PLA to make self-biodegradable plastic.

Plastic production reached 400 million tons in 2022 (ref. 1), with packaging and single-use plastics accounting for a substantial amount of this2. The resulting waste ends up in landfills, incineration or the environment, contributing to environmental pollution3. Shifting to biodegradable and compostable plastics is increasingly being considered as an efficient waste-management alternative4. Although polylactide (PLA) is the most widely used biosourced polymer5, its biodegradation rate under home-compost and soil conditions remains low6-8. Here we present a PLA-based plastic in which an optimized enzyme is embedded to ensure rapid biodegradation and compostability at room temperature, using a scalable industrial process. First, an 80-fold activity enhancement was achieved through structure-based rational engineering of a new hyperthermostable PLA hydrolase. Second, the enzyme was uniformly dispersed within the PLA matrix by means of a masterbatch-based melt extrusion process. The liquid enzyme formulation was incorporated in polycaprolactone, a low-melting-temperature polymer, through melt extrusion at 70 °C, forming an 'enzymated' polycaprolactone masterbatch. Masterbatch pellets were integrated into PLA by melt extrusion at 160 °C, producing an enzymated PLA film (0.02% w/w enzyme) that fully disintegrated under home-compost conditions within 20-24 weeks, meeting home-composting standards. The mechanical and degradation properties of the enzymated film were compatible with industrial packaging applications, and they remained intact during long-term storage. This innovative material not only opens new avenues for composters and biomethane production but also provides a feasible industrial solution for PLA degradation.

PPARG is dispensable for bovine embryo development up to tubular stages.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors (PPARs) mediate the signaling actions of prostaglandins and other lipids and, between them, PPARG has been pointed out to play a relevant role on conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG KO embryos in vitro by two independent gene ablation strategies and assess their developmental ability. In vitro development to Day (D) 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass and trophectoderm cell numbers were similar between WT and KO D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, and embryonic disc formation and hypoblast migration rates were unaffected by the ablation. The development to tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disc was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

HH5 double-carrier embryos fail to progress through early conceptus elongation.

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause preterm mortality. Holstein haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the transcription factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated with the inadvertent cross between carriers. To determine whether HH5 DC survive to maternal recognition of pregnancy, embryonic day (E) 14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. Double-carrier E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic reduction of their extra-embryonic membranes, which were >26-fold shorter than those of carrier or noncarrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos-functionally equivalent to DC embryos-were generated by clustered regularly interspaced short palindromic repeats (CRISPR) technology and cultured to the blastocyst stage, in vitro culture day (D) 8, and to the early embryonic disc stage, D12. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mitochondrial DNA (mtDNA) content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, which is required for pregnancy recognition.

PUF60-related developmental disorder: A case series and phenotypic analysis of 10 additional patients with monoallelic PUF60 variants.

PUF60-related developmental disorder (also referred to as Verheij syndrome), resulting from haploinsufficiency of PUF60, is associated with multiple congenital anomalies affecting a wide range of body systems. These anomalies include ophthalmic coloboma, and congenital anomalies of the heart, kidney, and musculoskeletal system. Behavioral and intellectual difficulties are also observed. While less common than other features associated with PUF60-related developmental disorder, for instance hearing impairment and short stature, identification of specific anomalies such as ophthalmic coloboma can aid with diagnostic identification given the limited spectrum of genes linked with this feature. We describe 10 patients with PUF60 gene variants, bringing the total number reported in the literature, to varying levels of details, to 56 patients. Patients were recruited both via locally based exome sequencing from international sites and from the DDD study in the United Kingdom. Eight of the variants reported were novel PUF60 variants. The addition of a further patient with a reported c449-457del variant to the existing literature highlights this as a recurrent variant. One variant was inherited from an affected parent. This is the first example in the literature of an inherited variant resulting in PUF60-related developmental disorder. Two patients (20%) were reported to have a renal anomaly consistent with 22% of cases in previously reported literature. Two patients received specialist endocrine treatment. More commonly observed were clinical features such as: cardiac anomalies (40%), ocular abnormalities (70%), intellectual disability (60%), and skeletal abnormalities (80%). Facial features did not demonstrate a recognizable gestalt. Of note, but remaining of unclear causality, we describe a single pediatric patient with pineoblastoma. We recommend that stature and pubertal progress should be monitored in PUF60-related developmental disorder with a low threshold for endocrine investigations as hormone therapy may be indicated. Our study reports an inherited case with PUF60-related developmental disorder which has important genetic counseling implications for families.

Effect of arachidonic acid on pre- and post-hatching in vitro bovine embryo development.

CONTEXT: Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. AIMS: To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro -produced bovine embryos. METHODS: Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333µM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100µM AA up to Day 12. KEY RESULTS: Pre-hatching development to blastocyst was completely abrogated at 333µM AA, whereas blastocyst rates and cell numbers were not altered at 100µM AA. Impaired post-hatching development was observed at 100µM AA, whereas no effect on survival rates was noted at 5, 10 and 20µM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20µM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5-10µM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos. CONCLUSIONS: Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development. IMPLICATIONS: AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages.

Post-fire management effects on hillslope-stream sediment connectivity in a Mediterranean forest ecosystem.

Forest fires intensify sediment transport and aggravate local and off-site consequences of soil erosion. This study evaluates the influence of post-fire measures on structural and functional sediment connectivity (SC) in five fire-affected Mediterranean catchments, which include 929 sub-catchments, by using the "aggregated index of connectivity" (AIC) at two temporal scenarios: I) immediately after the fire and before implementing post-fire practices ('Pre-man'), and II) two years after the fire ('Post-man'). The latter includes all the emergency stabilization practices, that are hillslope barriers, check-dams and afforestation. The stream system was set as the target of the computation (STR), to be representative of intense rainfall-runoff events with effective sediment delivery outside the catchments. Output normalization (AICN) allows comparing the results of the five basins between them. The sedimentological analysis is based on specific sediment yield (SSY) -measured at the check-dams installed after the fire -, and this data is used for output evaluation. Stream density and slope variables were the most influential factors on AICN-STR results at the sub-catchment scale. Post-fire hillslope treatments (barriers when built in high densities and afforestation) significantly reduced AICN-STR in comparison with untreated areas in both structural and functional approaches. Despite the presence of hillslope treatments, the higher erosive rainfall conditions resulted in higher AICN-STR values in the Post-man scenario (functional approach). A positive and good correlation was found between the measured SSY and the AICN-STR changes due to the post-fire practices and vegetation recovery, showing the good correspondence of the computation results and the real sediment dynamics of the studied catchments. Overall, AICN demonstrated to be a useful and versatile tool for post-fire management, which needs further research to optimize its applicability.

Cemiplimab in Advanced Cutaneous Squamous Cell Carcinoma: Real-World Experience in a Monographic Oncology Center.

Management of advanced cSCC is challenging, and many available systemic medications have modest efficacy. Cemiplimab has demonstrated efficacy in the treatment of advanced cSCC in clinical trials, but real-world data are still limited. With the objective of evaluating the efficacy of cemiplimab in a real-world clinical setting, we conducted a prospective observational study of 13 patients with advanced cSCC. Six patients (46%) had locally advanced disease, while 7 (54%) had metastatic disease. A total of 8 patients (62%) responded to cemiplimab. Five (38%) showed a partial response, while 3 (23%) showed a complete response. Four patients with an initial partial response presented subsequent disease progression during follow-up. Six patients (46%) developed AEs, most of which were mild (G1). PFS was 5.9 months, with a median follow-up was 9 months. In conclusion, cemiplimab demonstrated its utility in the treatment of advanced cSCC, with acceptable response rates, a remarkable number of complete responses, and a very good safety profile.

Cemiplimab in Advanced Cutaneous Squamous Cell Carcinoma: Real-World Experience in a Monographic Oncology Center. / [Artículo traducido] Cemiplimab en el carcinoma de células escamosas cutáneo avanzado: experiencia del mundo real en un centro oncológico monográfico.

Management of advanced cSCC is challenging, and many available systemic medications have modest efficacy. Cemiplimab has demonstrated efficacy in the treatment of advanced cSCC in clinical trials, but real-world data are still limited. With the objective of evaluating the efficacy of cemiplimab in a real-world clinical setting, we conducted a prospective observational study of 13 patients with advanced cSCC. Six patients (46%) had locally advanced disease, while 7 (54%) had metastatic disease. A total of 8 patients (62%) responded to cemiplimab. Five (38%) showed a partial response, while 3 (23%) showed a complete response. Four patients with an initial partial response presented subsequent disease progression during follow-up. Six patients (46%) developed AEs, most of which were mild (G1). PFS was 5.9 months, with a median follow-up was 9 months. In conclusion, cemiplimab demonstrated its utility in the treatment of advanced cSCC, with acceptable response rates, a remarkable number of complete responses, and a very good safety profile.

Burning season and vegetation coverage influenced the community-level physiological profile of Mediterranean mixed-mesogean pine forest soils.

Knowledge of forest soil ecology is necessary to assess vulnerability to disturbances, such as wildfires, and improve its microbial diversity and functional value. Soil microbiota play an important role in forest soil processes and are a key driver of postfire recovery, but they are very vulnerable to heat. According to future scenarios for climate and land-use change, fire regimes will undergo transformations in semiarid terrestrial ecosystems, mainly in the Mediterranean Basin. To develop tools for forest management in fire-prone areas, i.e., fire prevention, we assessed the impact of prescribed burnings on soil microorganisms in Mediterranean mixed pine forests. We hypothesised that low severity fire burns would not influence the functional diversity of soil microorganisms, although the burning season could influence that response due to seasonal variations in its vulnerability. We used the Biolog EcoPlate System to record soil biological indicators and assess the effect of the prescribed burning season (early or late season) on bacterial communities, including the soil-plant interphase. The soil microbiome response differed significantly according to vegetation coverage but prescribed burning season was not directly related. Burning increased the proportions of soil organic matter and soil organic carbon, and also promoted cation-exchange capacity and total phosphorus, which were higher following spring burns. Microbial richness and the Shannon-Weaver diversity index both showed a positive correlation with vegetation cover. However, microbial richness was triggered after burning uncovered patches of vegetation. We also noted differences in the usage pattern for the six substrate groups defined in our study: the use of carboxylic acids, amino acids and carbohydrates was higher in unburned plots and those subject to late burns, whereas amino acids did not predominate in early burn plots.

RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.

Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.

Changes in soil hydraulic conductivity after prescribed fires in Mediterranean pine forests.

Prescribed fire removes or reduces the plant material that is prone to forest fires by creating fuel discontinuity and minimising fire intensity. This forest management tool potentially impacts Mediterranean ecosystems hydrological response by influencing water infiltration into soil. As direct measurements (e.g. by infiltrometers) of unsaturated infiltration in soil subjected to prescribed fires are scarce, this study has evaluated changes in soil hydraulic conductivity (SHC) using Minidisk infiltrometer after prescribed fires in representative plots of forests in the Iberian Peninsula under Mediterranean semi-arid conditions: (i) pure forest of Black pine Arnold ssp salzmannii; (ii) mixed forest of Maritime and Black pine; (iii) mixed forest of Aleppo and Maritime pine. The results have shown that fire reduced the organic layer thickness and its organic matter content. Consequently, after the prescribed fire the water content of burned plots was always lower than in untreated soils; conversely, the reverse soil behaviour was noticed before applying fire. Compared to the untreated soils, and with very few exceptions, prescribed fire did not cause significant changes in SHC. No general patterns in the comparisons between treatments (burned/unburned soils), in time evolution after fires and in the interactions between these effects were detected. This means that the SHC of burned soils followed the temporal variations of untreated soils. The lack of significance of these differences between treatments could be due to the low-fire severity and the limited effect of temperature in the mineral layer on soil hydraulic properties. This effect was expected and agrees with other studies. Overall prescribed fires did not alter SHC in Mediterranean forest ecosystems under unsaturated conditions since fire was of low-severity.

Effects of post-fire hillslope stabilisation techniques on chemical, physico-chemical and microbiological soil properties in mediterranean forest ecosystems.

Postfire restoration practices encompass those which aim to reduce negative wildfire impacts and to improve burned area rehabilitation. Contour-felled log debris (CFD) and log erosion barriers (LEB) are two techniques used worldwide on hillslopes after wildfires in order to avoid soil erosion. In this context, it is essential to evaluate how these restoration techniques can affect soil properties by increasing or decreasing wildfire impacts. The effects on several physico-chemical and biological soil parameters were here investigated by comparing three differently treated post-fire zones. Three randomly 20 × 20 m distributed plots were set up five years after wildfire in the burned and contour-felled log debris areas (CFD plots), three others in the burned and log erosion barriers area (LEB plots) and three others in the burned and unmanaged plots (BNa plots). Three more plots were set up in an unburned area close to the burned area (UB plots). The results revealed that LEB and, to a lesser extent CFD, improved postfire soil quality, which a priori favoured helped the recovery of ecosystem functions. Our results also indicate greater efficacy of LEB and CFD in retaining sediments by limiting loss of nutrients, which is considered essential to recover vegetation after a wildfire. Post-fire restoration plans should consider the use of LEB and CFD when aiming to favour ecosystem recovery processes after wildfires.

Multi-faceted Characterization of Wet-milled Griseofulvin Nanosuspensions for Elucidation of Aggregation State and Stabilization Mechanisms.

Characterization of wet-milled drug suspensions containing neutral polymer-anionic surfactant as stabilizers poses unique challenges in terms of assessing the aggregation state and examining the stabilization mechanisms. Using a multi-faceted characterization method, this study aims to assess the aggregation state of wet-milled griseofulvin (GF) nanosuspensions and elucidate the stabilization mechanisms and impact of stabilizers. Two grades, SSL and L, of hydroxypropyl cellulose (HPC) with molecular weights of 40 and 140 kg/mol, respectively, were used as a neutral stabilizer at concentrations varying from 0 to 7.5% (w/w) without and with 0.05% (w/w) sodium dodecyl sulfate (SDS). The aggregation state was examined via laser diffraction, scanning electron microscope (SEM) imaging, and rheometry. Zeta potential, stabilizer adsorption, surface tension, and drug wettability were used to elucidate the stabilization mechanisms. The results suggest that deviation from a uni-modal PSD and pronounced pseudoplasticity with power-law index lower than one signify severe aggregation. Polymer or surfactant alone was not able to prevent GF nanoparticle aggregation, whereas HPC-SDS combination led to synergistic stabilization. The effect of polymer concentration was explained mainly by the stabilizer adsorption and partly by surface tension. The synergistic stabilization afforded by HPC-SDS, traditionally explained by electrosteric mechanism, was attributed to steric stabilization provided by HPC and enhanced GF wettability/reduced surface tension provided by SDS. Zeta potential results could not explain the mitigation of aggregation by HPC-SDS. Overall, this study has demonstrated that the elucidation of the complex effects of HPC-SDS on GF nanosuspension stability entails a multi-faceted and comprehensive characterization approach.

Adsorptive process design for the separation of hexane isomers using zeolites.

The product of catalytic isomerization is a mixture of linear and branched hydrocarbons that are in thermodynamic equilibrium, and their separation becomes necessary in the petrochemical industry. Zeolite 5A is usually industrially used to sieve alkane isomers, but its pore size allows only the separation of linear alkanes from the monobranched and dibranched alkanes by a kinetic mechanism. A more efficient approach to improve the average research octane number would be to adsorptively separate the di-methyl alkanes as products and recycle both the linear and mono-methyl alkanes to the isomerization reactor. Since the microscopic processes of adsorbates in zeolites are generally difficult or impossible to determine by experiments, especially in the case of mixtures, molecular simulation represents an attractive alternative. In this computational study, we propose a conceptual separation process for hexane isomers consisting of several adsorptive steps. Different zeolite topologies were examined for their ability to conduct this separation based on adsorption equilibrium and kinetics.

CRISPR is knocking on barn door.

Genome modification at specific loci in livestock species was only achievable by performing homologous recombination in somatic cells followed by somatic cell nuclear transfer. The difficulty and inefficiency of this method have slowed down the multiple applications of genome modification in farm animals. The discovery of site-specific endonucleases has provided a different and more direct route for targeted mutagenesis, as these enzymes allow the ablation (KO) or insertion (KI) of specific genomic sequences on a single step, directly applied to zygotes. Clustered regularly interspaced short palindromic repeats (CRISPR), the last site-specific endonuclease to be developed, is a RNA-guided endonuclease, easy to engineer and direct to a given target site. This technology has been successfully applied to rabbits, swine, goats, sheep and cattle, situating genome editing in livestock species at an attainable distance, thereby empowering scientist to develop a myriad of applications. Genetically modified livestock animals can be used as biomodels to study human or livestock physiology and disease, as bioreactors to produce complex proteins, or as organ donors for transplantation. Specifically on livestock production, genome editing in farm animals may serve to improve productive genetic traits, to improve various animal products, to confer resistance to diseases or to minimize the environmental impact on farming. In this review, we provide an overview of the current methods for site-specific genome modification in livestock species, discuss potential and already developed applications of genome edition in farm animals and debate about the possibilities for approval of products derived from gene-edited animals for human consumption.

TLD and OSLD dosimetry systems for remote audits of radiotherapy external beam calibration.

The Imaging and Radiation Oncology Core QA Center in Houston (IROC-H) performs remote dosimetry audits of more than 20,000 megavoltage photon and electron beams each year. Both a thermoluminescent dosimeter (TLD-100) and optically stimulated luminescent dosimeter (OSLD; nanoDot) system are commissioned for this task, with the OSLD system being predominant due to the more time-efficient read-out process. The measurement apparatus includes 3 TLD or 2 OSLD in an acrylic mini-phantom, which are irradiated by the institution under reference geometry. Dosimetry systems are calibrated based on the signal-to-dose conversion established with reference dosimeters irradiated in a Co-60 beam, using a reference dose of 300 cGy for TLD and 100 cGy for OSLD. The uncertainty in the dose determination is 1.3% for TLD and 1.6% for OSLD at the one sigma level. This accuracy allows for a tolerance of ±5% to be used.

Using synoptic tracer surveys to assess runoff sources in an Andean headwater catchment in central Chile.

Headwater catchments in the Andes provide critical sources of water for downstream areas with large agricultural communities dependent upon irrigation. Data from such remote headwater catchments are sparse, and there is limited understanding of their hydrological function to guide sustainable water management. Here, we present the findings of repeat synoptic tracer surveys as rapid appraisal tools to understand dominant hydrological flow paths in the semi-arid Rio Grande basin, a 572-km2 headwater tributary of the 11,696-km2 Limarí basin in central Chile. Stable isotopes in stream water show a typical altitudinal effect, with downstream enrichment in δ2H and δ18O ratios. Seasonal signals are displayed in the isotopic composition of the springtime melting season water line with a steeper gradient, whilst evaporative effects are represented by lower seasonal gradients for autumn and summer. Concentrations of solutes indexed by electrical conductivity indicate that there are limited contributions of deeper mineralised groundwater to streamflow and that weathering rates vary in the different sub-catchments. Although simplistic, the insights gained from the study could be used to inform the structure and parameterisation of rainfall runoff models to provide seasonal discharge predictions as an evidence base for decision making in local water management.

Modulation of autoimmune arthritis severity in mice by apolipoprotein E (ApoE) and cholesterol.

Apolipoprotein E (ApoE) deficiency promoted an exacerbation of autoimmune arthritis in mice by inducing proinflammatory immune responses. In this study we analysed the contribution of hypercholesterolaemia and/or the absence of ApoE anti-inflammatory properties, unrelated to its function in the control of cholesterol metabolism, towards the acceleration of arthritis in these mutant animals. The induction and severity of collagen type II-induced arthritis (CIA) were compared for B10.RIII wild-type (WT), B10.RIII.ApoE+/- , B10.RIII.ApoE-/- and B10.RIII.low-density lipoprotein receptor (LDLR-/- ) mice with different concentrations of circulating ApoE and cholesterol. A 50-70% reduction in serum levels of ApoE was observed in heterozygous B10.RIII.ApoE+/- mice in comparison to B10.RIII.WT, although both strains of mice exhibited similar circulating lipid profiles. This ApoE reduction was associated with an increased CIA severity that remained lower than in homozygous B10.RIII.ApoE-/- mice. An important rise in circulating ApoE concentration was observed in hypercholesterolaemic B10.RIII.LDLR-/- mice fed with a normal chow diet, and both parameters increased further with an atherogenic hypercholesterolaemic diet. However, the severity of CIA in B10.RIII.LDLR-/- mice was similar to that of B10.RIII.WT controls. In conclusion, by comparing the evolution of CIA between several strains of mutant mice with different levels of serum ApoE and cholesterol, our results demonstrate that both hypercholesterolaemia and ApoE regulate the intensity of in-vivo systemic autoimmune responses.

Spermatozoa telomeres determine telomere length in early embryos and offspring.

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.