Chemical modifications of RNA molecules, collectively referred to as epitranscriptomics, have emerged as a critical layer of post-transcriptional gene regulation. Among these modifications, N6-methyladenosine (m6A) plays a key role in modulating RNA stability, localization, and translational efficiency without altering the underlying nucleotide sequence. In recent years, epitranscriptomics has been increasingly recognized for its involvement in mammalian reproductive processes. In mammalian oocytes, which undergo an extended period of transcriptional quiescence and rely predominantly on post-transcriptional regulation of maternally stored transcripts, RNA modifications are essential for proper gene expression control. During oocyte maturation and early pre-implantation embryo development, epitranscriptomic mechanisms ensure the timely turnover and translational regulation of maternal mRNAs, thereby supporting the correct execution of the maternal-to-zygotic transition. The proper accomplishment of these processes is crucial for the acquisition of developmental competence. Reversible RNA modifications therefore add an additional layer of complexity to the regulation of gene expression in oocytes and early embryos. This mini-review summarizes current knowledge on the role of m6A RNA methylation in mammalian oocyte developmental competence and early embryogenesis, highlighting recent experimental evidence and discussing potential implications for fertility and assisted reproductive technologies.

We hypothesized that in dairy cattle maternal energy restriction applied during two gestational windows (up to day 80 or 120 of gestation) impairs ovarian and cardiovascular development in juvenile female offspring. We also investigated the role of maternal leptin and testosterone in developmental programming in calves. Holstein-Friesian heifers were randomly assigned to one of three experimental groups; starting 10 days before artificial insemination, they were individually fed at (i) 0.6 of their maintenance energy requirements (M) up to day 80 (Nutrient Restricted, NR80) or (ii) day 120 of gestation (NR120); (iii) 1.8 M until day 120 of pregnancy (Control). Plasma leptin concentrations increased transiently in nutritionally restricted heifers pregnant with a single female calf, but maternal testosterone concentrations were not influenced by diet. Calves had similar body growth, but daughters of NR80 and NR120 had impaired ovarian development, as assessed by reduced gonadal weight, fewer surface antral and primary follicles, and recovered cumulus-oocyte complexes, as well as lower circulating anti-Müllerian hormone concentrations. Cardiovascular morphology and function in the offspring were not influenced by maternal diet, as determined by peripheral arterial blood pressure, echocardiography, post-mortem heart weight, and aortic circumference. Regardless of its duration (until day 80 or 120 of gestation), nutritional restriction resulted in a similar alteration of ovarian development in juvenile progeny, but cardiovascular development was unaltered. Evidence suggests that the window of development that encompasses the peri-ovulatory period to the first 2.6 months of gestation is critical in ovarian programming and that maternal leptin may be involved.

Heat stress affects fertility and productivity in dairy cattle, yet field studies from the Mediterranean area remain limited. This retrospective, observational cohort study aimed to investigate seasonal variation in: (1) conception and pregnancy rates (at 30 and 60d post-AI, respectively), number of services per conception, submission rate, calving to conception interval, and gestation length; (2) milk production and composition in Holstein Friesian cattle in two Italian farms during three consecutive years. Reproductive data, milk production, and composition were analyzed both monthly and based on three categories: Minimum risk temperature-humidity index (THI) < 56, Low risk (56 < THI < 68), and High risk (THI> 68). High, Low, and Minimum risk conditions occurred for approximately 4 months/year. In heifers that were not exposed to cooling system, the monthly submission rate peaked in May (17.9%) and was the lowest in September (9.4% P < 0.001); also, a greater submission rate was recorded under Minimum and Low risk compared to High risk (P < 0.001). In lactating cows which were exposed to cooling systems when THI exceeded 68, conception rate at 30d post-AI, pregnancy rate at 60d post-AI, number of services per conception, monthly submission rate were influenced by month (P < 0.001). Under High risk conception rate at 30d post-AI (High risk = 37.2%; Minimum risk = 55.2%; P < 0.05), pregnancy rate at 60d post-AI (High risk = 29.4%, Minimum risk = 45.8%; P < 0.05) and submission rate (High risk = 13.1%, Minimum risk = 17.9%; P < 0.001) were reduced compared with Minimum risk. Milk production peaked in spring and decreased in summer and was lower under High (10.90 ton/day) than Minimum risk (11.91 ton/day; P < 0.01). When THI increased, milk fat, protein and lactose percentages decreased, whereas somatic cell count and milk urea nitrogen increased (P < 0.05). Evidence indicates that cattle in the Mediterranean region can be exposed to mean THI ≥ 68 for four consecutive months; high environmental temperatures may decrease conception rates by nearly 20% and impair milk production in cows.

Cell spheroids are widely studied for their potential applications in tissue engineering and regenerative medicine. The present work investigated the effects of cryopreservation on spheroids derived from ovine fibroblasts, depending on spheroid size (140 or 220 µm). Specifically, it explored how cryopreservation impacted several biological and physical parameters including cell damage, viability, metabolism, adhesion, proliferation, and spheroid mass density, weight, and diameter at three time points after thawing. A Live/Dead assay provided a visual assessment of cell damage, cell viability and metabolic activity were assessed by an Alamar Blue assay, and a replating assay evaluated cell adhesion and proliferation capabilities. Spheroid mass density, weight, and diameter were quantified by the W8 Biophysical Analyzer, creating accurate biophysical profiles. Real-time PCR (RT-PCR) analysis was employed to uncover gene expression changes following cryopreservation. Our findings indicate that spheroids measuring 140 µm in diameter largely maintained their biophysical features and cell viability post-cryopreservation, whereas those at 220 µm exhibited a decline in both vitality and mass density. The reduced vitality of 220 µm spheroids likely reflects size-related limitations in cryoprotectant diffusion and stress within the core. Overall, this study provides a comprehensive understanding of how cryopreservation affects ovine fibroblast spheroid biophysics and cellular integrity, laying the groundwork for improved preservation techniques for cell spheroids.

N6-methyladenosine (m6A) is the most prevalent base epigenetic modification within eukaryotic mRNAs. It participates in post-transcriptional regulation, including maternal RNA maintenance and decay in mouse oocytes and during maternal-to-zygotic transition. The landscape in other mammalian species remains largely unexplored. The present work analyzed m6A dynamics in sheep cumulus oocyte complexes (COCs), during in vitro maturation. To explore potential relationships with oocyte developmental competence, a previously established model consisting of oocytes derived from adult and prepubertal sheep was adopted. m6a dynamics were analyzed in terms of m6A RNA methylation abundance in cumulus cells (CCs) by colorimetric assay and expression of key m6A methylation-related proteins (METTL3, METTL14, METTL16, VIRMA, YTHDC1, YTHDC2, YTHDF2, YTHDF3, ALKBH5, and FTO) in both cumulus cells and oocytes by real-time PCR. We report the dynamics of m6A in sheep COCs, and reveal alterations in both oocytes and cumulus cells derived from prepubertal donors. These changes were observed in terms of m6A RNA methylation levels and transcript dynamics of several m6A methylation-related proteins. Notably, our study shows that dysregulations occur after IVM. Overall, this work describes for the first time the dynamics of m6A in sheep COCs and uncovers the involvement of m6A RNA methylation in oocyte developmental potential.

The cell cortex, a cytoskeletal network with regulatory signalling pathways, is localized beneath the cell membrane: it is especially prominent in mammalian oocytes. As in other cells, the cortex ensures appropriate shape and robustness of oocytes. It is also involved in other key and more specific functions. The cortex is part of the interface between the germinal and somatic cell compartments; as such, it participates in the delicate bi-directional interaction by which oocytes regulate cumulus cell function and in return, oocytes receive nutrients and regulative factors from cumulus cells. During oocyte maturation, fertilization, and early development, the cortex undergoes major structural and functional modifications. Such changes are needed to support crucial processes, including meiotic spindle localization, polar body extrusion, chromosome segregation, and pronuclear formation. Cortex dysregulation may be also implicated in blastomere fragmentation during early embryo development. Mechanical properties of the cortex are associated with oocyte quality and developmental competence; with appropriate technology, such properties could be harnessed to develop new approaches to non-invasive oocyte assessment in human IVF.

To investigate the effects of microplastics (MPs) on the human endometrium in vitro. A predictive 3D endometrial in vitro model was generated using highly porous scaffolds where human endometrial stromal (hESC) and epithelial (hEEC) cells were co-cultured for 35 days. The newly generated endometrial barrier was then exposed to different MP concentrations (from 0.25 to 50 mg/ml) for 24 h and 48 h, respectively. Histological staining and functional analyses were performed to assess the endometrial barrier integrity. Molecular studies and collagen deposition were evaluated to investigate the possible activation of pro-apoptotic and pro-fibrotic related pathways. MP exposure for 24 h does not affect endometrial barrier integrity nor collagen synthesis and deposition. Similar responses are detected when concentrations between 0.25 and 1 mg/ml are used for 48 h. In contrast, 48-h incubations with higher doses (10–50 mg/ml MPs) induce epithelial barrier alterations, reduce TEER values and decrease ZO1 and CDH1 gene transcription. This is accompanied by the activation of pro-fibrotic signalling pathways resulting in collagen increment, which often accompanies endometriosis-related alterations. The data obtained suggest MP ability to exert deleterious effects in vitro on human endometrium, with a possible negative impact on its functionality and receptivity.

Application: High-fertility timed-AI (TAI) protocols facilitates the application of sexed semen in Holstein heifers.Introduction: The widespread use of sexed semen requires the development of TAI programs with high fertility (Bo et al., 2018).Two experiments were designed to evaluate of the length of insertion of the progesterone (P4) device on follicular characteristics and P/AI in Holstein Heifers treated with the oestradiol/P4 based protocol, named J-Synch, and inseminated with sexed semen.Materials and Methods: Holstein heifers (n = 14 in Experiment 1 and 336 in Experiment 2) were used.On Day 0, all heifers received 2 mg oestradiol benzoate (Over, Argentina) and a device containing 0.7 g P4 (Sincrover, Over).The P4 device was removed on Day 6 in the 6-d J-Synch group and on Day 7 in the 7-d J-Synch group.All heifers received 150 lg D(+) cloprostenol (Prostal, Over) at device removal and were tail painted for oestrus detection.In Experiment 1, heifers were scanned twice daily from device removal to ovulation.In Experiment 2, heifers with >30% of the tail-paint rubbed off by 72 h after device removal were inseminated at that time, whereas those without the tail-paint rubbed-off received 10 lg buserelin (Gestar, Over) and were inseminated 12 h later.All heifers in Experiment 2 were inseminated with sexed semen from 6 bulls (Sexcel, ABS, USA) that were equally distributed among groups and were examined for pregnancy 30 days after AI.Data were analysed using ANOVA in Experiment 1 and GLM mixed procedure for binary data with a logit link in Experiment 2. Results: In Experiment 1, the interval from device removal to ovulation tended (P = 0.08) to be longer in the 6-d J-Synch group (96.0 ± 5.8 h) than in the 7-d J-Synch group (82.5 ± 5.0 h).The diameter of the largest follicle at the time of device removal and before ovulation did not differ among groups (8.1 ± 1.5 and 13.7 ± 0.7 mm vs 10.1 ± 1.5 and 1.9 ± 0.6 mm, for the 6 and 7-d J-Synch groups, respectively).In Experiment 2, although oestrus expression did not differ (86.4%, 146/1169 vs 87.4%, 146/167 for the 6 and 7-d J-Synch, respectively), P/AI was greater (P < 0.05) in those in the 7-d J-Synch (49.1%, 82/167) than those in the 6-d J-Synch group (37.9%, 64/169).Conclusions: Delaying the removal of the P4 device by one day in the J-Synch protocol resulted in higher P/AI in Holstein heifers inseminated with sexed semen.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown–rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo–cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

The age‐associated decline in female fertility is largely ascribable to a decrease in oocyte quality. This phenomenon is multifaceted and influenced by numerous interconnected maternal and environmental factors. An increase in the rate of meiotic errors is the major cause of the decline in oocyte developmental competence. However, abnormalities in the ooplasm accumulating with age — including altered metabolism, organelle dysfunction, and aberrant gene regulation — progressively undermine oocyte quality. Stockpiling of maternal macromolecules during folliculogenesis is crucial, as oocyte competence to achieve maturation, fertilization, and the earliest phases of embryo development occur in absence of transcription. At the same time, crucial remodeling of oocyte epigenetics during oogenesis is potentially exposed to interfering factors, such as assisted reproduction technologies (ARTs) or environmental changes, whose impact may be enhanced by reproductive aging. As the effects of maternal aging on molecular mechanisms governing the function of the human oocyte remain poorly understood, studies in animal models are essential to deepen current understanding, with translational implications for human ARTs. The present mini review aims at offering an updated and consistent view of cytoplasmic alterations occurring in oocytes during aging, focusing particularly on gene and epigenetic regulation. Appreciation of these mechanisms could inspire solutions to mitigate/control the phenomenon, and thus benefit modern ARTs.

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Cryopreservation is a fundamental procedure to preserve the structure and function of cells and tissues by storing them at low temperatures for long periods [...].

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Human activities are having increasingly devastating effects on the health of marine and terrestrial ecosystems. Studying the adaptive responses of animal species to changes in their habitat can be useful in mitigating this impact. Vultures represent one of the most virtuous examples of adaptation to human-induced environmental changes. Once dependent on wild ungulate populations, these birds have adapted to the epochal change resulting from the birth of agriculture and livestock domestication, maintaining their essential role as ecological scavengers. In this review, we retrace the main splitting events characterising the vultures' evolution, with particular emphasis on the Eurasian griffon Gyps fulvus. We summarise the main ecological and behavioural traits of this species, highlighting its vulnerability to elements introduced into the habitat by humans. We collected the genetic information available to date, underlining their importance for improving the management of this species, as an essential tool to support restocking practices and to protect the genetic integrity of G. fulvus. Finally, we examine the difficulties in implementing a coordination system that allows genetic information to be effectively transferred into management programs. Until a linking network is established between scientific research and management practices, the risk of losing important wildlife resources remains high.

Heat stress (HS) is characterised by an elevation in body temperature that ultimately undermines organism physiology. Most livestock production occurs in tropical regions under potential HS conditions that diminish productive and reproductive potential. Despite extensive evidence of HS-mediated effects in cell function, stage-specific detrimental effects of HS during oogenesis remain elusive. Mouse models represent an attractive alternative for faster interrogation of stage-specific phenomena during oogenesis. Therefore, the aim of the study was to determine the effects of HS exposure during the major window of female mice germ-cell DNA methylation programming. CD1/Swiss female mice with litters (F0 progeny) at postnatal Day 10 (P10) were randomly allocated to HS (35°C/12-h light; 21°C/12-h dark) or control (CTL: 21°C/24h) for 11 days. The F0 progeny were weaned at P21 and superovulated after reaching puberty at P35. F0 females were superovulated by intraperitoneal injections with 5.0IU of equine chorionic gonadotrophin (PMSG) followed by 5.0IU of human chorionic gonadotrophin (hCG) within a 48-h interval. Pre-implantation embryos were harvested at Day E3.5 in M2 medium under a stereomicroscope. One F0 female per litter was randomly mated to control mice when it reached 6 weeks of age. Data were subjected to least-squares analysis of variance using the General Linear Models procedure of SAS (SAS Institute Inc.). The experiment was replicated twice (CTL: n=4 F0 females and HS: n=4 F0 females). Preliminary results are given as LSM±s.e.m. There was no effect of heat stress on the number of embryos collected per female (CTL: 9.75±4.87 vs. HS: 11.25±4.81) or the percentage of non-viable embryos (CTL: 25.0±0.23% vs. HS: 42.5±0.25%). However, heat stress tended (P=0.07) to reduce the percentage of embryos that reached the morula stage from 63.5±0.08% for CTL to 35.1±0.09% for HS. The percentage of blastocysts collected (CTL: 11.45±0.18% vs. HS: 22.32±0.19%) and litter size of F0 females (CTL: 7.47±1.76 vs. HS: 7.66±1.47) was not affected by treatment. In conclusion, exposure of female mice to 11-day HS during the major wave of de novo DNA methylation during oocyte growth tended to reduce subsequent pre-implantation embryonic development, although it did not affect full-term development after natural mating.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.