Abstract Background The ovarian reserve, primarily composed of primordial (PMF), primary (PF), and secondary follicles (SF), holds great potential for fertility preservation, yet remains inaccessible mainly due to the challenges of replicating early folliculogenesis in vitro. The molecular mechanisms governing the activation and differentiation of oocytes from the dormant PMF pool remain poorly defined. In this study, we used a bovine model to investigate early follicular development. Results The transcriptome of isolated PMF, PF, and SF revealed different profiles of the follicles during early folliculogenesis. Differential gene expression analyses between PF versus PMF and SF versus PF revealed 689 and 3,206 significantly regulated genes, respectively (FDR < 0.05), highlighting key pathways including PI3K-Akt, Wnt, mTOR, and ECM-receptor interaction. Between PF versus PMF and SF versus PF, 13 and 69 DEGs were identified as transcription factors, respectively. A Likelihood Ratio Test followed by hierarchical clustering identified four major gene expression clusters across the three stages, showing significant enrichment in processes such as ECM-related functions and cell cycle. Based on the enrichment of the cell cycle pathway, we focused on RAD51, which was highly expressed in preantral follicles. Immunohistochemical analysis further showed that RAD51 protein localized in the ooplasm, along with the mitochondrial marker HSP60. Conclusion This is the first study to perform bulk RNA sequencing on isolated preantral follicles (PMF, PF, and SF) in cattle. Our findings provide new insights into the stage-specific mechanisms regulating follicle activation and growth in cattle, laying the groundwork for future in vitro fertility preservation strategies in both clinical and conservation contexts.

IntroductionDairy farming has a preferential demand for female calves to sustain milk production. Spermatozoa carrying the X chromosome, selected based on the higher content in DNA, allow to obtain almost exclusively female offspring. However, the separation procedures are stressful to the cells and induce mechanical and chemical modifications that negatively impact the survival, motility, and ultimately the fertilization potential. AIM: Considering that overall the X-sorted spermatozoa are characterized by compromised membrane integrity and premature capacitation, a series of experiments was conducted to test some optimizations of the in vitro fertilization (IVF) procedures to increase the embryo yield using commercially available X-sorted semen.MethodsThree series of experiments were conducted that led us to progressively refine the IVF procedure with X-sorted spermatozoa. First different protocols for semen processing were assessed for the yield in motile spermatozoa. Then the most promising were used in an IVF setting to investigate the fertilization ability. Finally the most performant protocol was used to investigate the effect of different IVF co-incubation times on the embryo yield.ResultsBy modifying some parameters of the discontinuous density gradient centrifugation such as the volume of the gradient, the proportion of PVP-coated colloidal silica solution, the centrifugation speed and the time of processing, along with the duration of the IVF culture, higher motility and fertilization and blastocyst rates were obtained (P<0.05).DiscussionOverall, while studies indicate that different bulls may require specific conditions for optimal fertilization and embryo yield, the present report shows that small adjustments to semen processing can significantly improve the efficiency of the in vitro embryo production with X-sorted semen.

The ability to grow undifferentiated oocytes in vitro from primordial follicles would increase the availability of fully grown oocytes in fertility preservation programs and other downstream applications. To date, the development of living offspring in vitro from the primordial follicle reserve has only been achieved in mice, proving the principle of the potential value of follicle culture as a source of competent oocytes. In certain pathophysiological conditions, such as polycystic ovarian syndrome, premature ovarian failure, or ovarian and blood cancer, where the ovarian tissue cannot be reintroduced into the patient, it is essential to isolate these follicles from the surrounding tissue and culture them in vitro. However, the culture systems that produce mature oocytes from isolated primordial follicles are still under investigation. Upon isolation from the ovarian microenvironment, a critical limiting factor is follicle death after a short period of culture. Previous studies suggest that glycine, a key component of glutathione (GSH), plays a protective role against the programmed cell death mechanism, ferroptosis, in in vitro matured porcine oocytes via the System Xc-/GSH/glutathione peroxidase 4 (GPX4) axis. Employing a previously developed high-yielding primordial follicle mechanical isolation strategy and a defined culture system, we used RNA-seq to advance the knowledge of the main transcriptional events and molecular factors determining follicle fate in a 2D culture system. Our transcriptome analyses identified genes involved in ferroptosis that may bring about primordial follicle death. To suppress ferroptosis, glycine supplementation maintained the viability of primordial follicles at ∼85% for 16 h. Future improvements to the culture system should inhibit programmed cell death mechanisms and ensure the physiological compliance of the genes regulating primordial follicle activation and transition to the primary stage, along with effective supplementation media to develop isolated primordial follicles in vitro.

Unlocking the developmental potential of oocytes at various stages of folliculogenesis represents a major challenge in reproductive biology and assisted reproductive technologies. While in vitro maturation (IVM) of fully grown oocytes is widely applied, the vast majority of oocytes enclosed within early-stage follicles remain underutilized. This review outlines current advancements in in vitro culture systems designed to support oocyte growth and differentiation, with particular attention to the contributions of the authors. Key developments, mainly encompassing the bovine species, include the use of prematuration strategies to enhance the competence of oocytes retrieved from antral follicles, stepwise in vitro culture protocols for growing oocytes from early antral follicles, and efforts to establish defined systems for preantral follicle culture. Emerging insights into chromatin dynamics, cumulus-oocyte communication, and epigenetic regulation are shaping the design of tailored culture environments. Despite promising progress, significant challenges remain in replicating the complexity of in vivo folliculogenesis, particularly in non-rodent models. Addressing these challenges will be critical to expanding the oocyte pool available for reproductive and biotechnological applications, with broad implications for fertility preservation, livestock breeding, and fundamental research.

Histone acetylation is an epigenetic modification responsible for changes in chromatin architecture, accessibility, and ultimately gene expression. At the onset of a new life, when the fully differentiated parental genomes fuse together to generate a new totipotent cell, the gametes' epigenetic program must be erased, and new ones are progressively installed. Together with other epigenetic modifications, histone acetylation participates in the early events of embryogenesis, undergoing dynamic changes that involve several amino acid residues on different histone proteins. By analyzing studies that followed these changes during the preimplantation development in different mammals, we identified critical windows of acetylation/deacetylation in relation to the oocyte-to-zygote transition, the activation of the embryonic genome, and the specification of cell lineages, all crucial events for early embryo development, the establishment of pluripotent embryonic tissue, and ultimately of a multicellular organism.Finally, this survey points out the possibility that while contributing to the necessary plasticity of the embryonic stem cells, the reversibility of histone acetylation/deacetylation patterns renders this mechanism prone to be hijacked by environmental conditions, such as maternal diet or pollutants, leading to the alterations of epigenetic marks that can be potentially transmitted to the daughter cells and up to adulthood.

Analyzing cellular health and metabolism without compromising cell integrity is a major challenge. We present a noninvasive technique using micro magnetic resonance spectroscopy (micro MRS) for nondestructive metabolic fingerprinting at the single-cell scale. This is an application of micro MRS to bovine preimplantation embryos (~8 cells) and oocytes (single cell), with measurements performed on a total of over 150 samples. Among various applications, this method holds significant potential for assisted reproductive technologies (ART), where metabolic assessments of preimplantation embryos could improve treatment outcomes. Early results indicate that classification models using micro MRS data effectively distinguish embryos with high developmental potential and show correlation with oocytes maturity. Furthermore, a multigenerational safety study in a mouse model revealed no adverse effects from embryo exposure to static magnetic field. These findings indicate that micro MRS is a promising, safe tool for assessing embryo metabolism, potentially improving the efficiency and outcomes of ART.

The ability to develop oocytes from the earliest follicular stages through maturation and fertilization in vitro would revolutionize fertility preservation in human medicine and animal breeding. Instead, current assisted reproductive technologies rely only on a limited portion of the female gamete reserve, corresponding to the antral population, while the preantral follicle reserve remains unexploited, mainly due to a lack of knowledge regarding the mechanisms that guide preantral follicle differentiation and folliculogenesis in vitro. This review highlights the efforts made thus far and suggests an approach to studying the mechanisms and ovarian environment to enhance preantral follicle culture systems.

Intracytoplasmic spermatid injection into oocytes has limited efficiency in cattle, with no offspring generated so far, partly due to ambiguous spermatid identification. This study aimed to develop and validate a method for isolating and characterizing bovine spermatids to improve the efficiency of spermatid intracytoplasmic injection. First, we optimized a protocol for spermatid isolation from bull testis using a discontinuous Percoll gradient and 10 μm mesh cell strainers. Next, we established a stage-specific separation strategy based on DNA content, size, and granularity using flow cytometry to distinguish round and elongating/elongated spermatids suitable for molecular analysis. Morphological assessment confirmed that 72.5% of isolated cells were at the spermatid stage, supported by a high haploidy rate, spermatid-specific transcript expression (PRM1, PRM2, SPACA9, SPERT), and SPERT protein detection. Viability assays showed that spermatids maintained intact DNA at 0 and 24 h at 4°C and 37°C, though mitochondrial activity and ROS levels increased over time, suggesting oxidative stress. When spermatids were injected into oocytes (n = 82), only 13.4% formed two pronuclei, whereas 46.3% exhibited a single pronucleus and a condensed chromatin spot, indicating incomplete activation or fertilization failure. This work contributes to refining bovine intracytoplasmic injection protocols. Future applications of this approach, particularly if functional spermatids can be derived from spermatogonia or embryonic cells, could help shorten the generational interval in cattle breeding.

In brief Preantral follicles constitute the largest follicle reserve in the mammalian ovary. This study assesses a mechanical isolation method to maximize the number of follicles retrieved from a defined cortex volume. Abstract Primordial, primary, and secondary follicles (collectively defined as preantral follicles) constitute the most abundant source of gametes inside the mammalian ovarian cortex. The massive isolation of preantral follicles and the refinement of stage-specific protocols for in vitro follicle growth would provide a powerful tool to boost the rescue and restoration of fertility in assisted reproduction interventions in human medicine, animal breeding, and vulnerable species preservation. Nevertheless, together with an efficient culture system, the most significant limitation to implementing in vitro follicle growth is the lack of an efficient method to isolate viable and homogeneous subpopulations of primordial, primary, and secondary follicles suitable for in vitro culture. Our study provides a strategy for high-yielding mechanical isolation of primordial, primary, and early secondary follicles from a limited portion of the ovarian cortex in the bovine animal model. In the first part of the study, we refined a mechanical isolation protocol of preantral follicles, adopting specific methodological strategies to separate viable and distinct subpopulations of primordial (oblate and prolate forms), primary, and early secondary follicles from 0.16 cm 3 of the ovarian cortex. In the second part of the study, we tested the effectiveness of the isolation protocol, considering the individual’s age as a critical factor, bearing in mind the progressive decrease in the ovarian reserve that naturally accompanies the reproductive life span. Our study provides a way for designing quantitative and conservative fertility preservation approaches to preserve organ function and minimize the invasiveness of the interventions, also considering age-related differences.

The mammalian ovary is a substantial source of oocytes arranged into follicles at various stages of folliculogenesis, from the primordial to the ovulatory ones. Primordial follicles constitute the most abundant source of gametes inside the mammalian ovary at any given time.The isolation of a high number of primordial follicles, together with the development of protocols for in vitro follicle growth, would provide a powerful tool to fully exploit the female reproductive potential and boost the rescue and restoration of fertility in assisted reproduction technologies in human medicine, animal breeding, and preservation of threatened species. However, the most significant limitation is the lack of efficient methods for isolating a healthy and homogeneous population of viable primordial follicles suitable for in vitro culture. Here, we provide a fast and high-yield strategy for the mechanical isolation of primordial follicles from limited portions of the ovarian cortex in the bovine animal model.

Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow’s genetic background had a higher impact than the oocyte’s maturation stage. Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.

The use of Assisted Reproductive Technologies (ART) is specifically recommended by FAO for livestock breeds classified as “endangered” or “vulnerable” concerning extinction risk. However, the success of conservation strategies implying ART depends on their acceptance among breeders. The present research explores farmers’ acceptability of ART for conserving the Varzese-Ottonese-Tortonese (VOT) cattle. Data have been collected through in-depth interviews with the cattle breeders members of the VOT Association. Results from this study are especially important considering the limited adoption and diffusion of ART among endangered livestock farmers and give a hint to support breeders in developing more economically sustainable conservation strategies.

Female fertility preservation via complete in vitro folliculogenesis is still chimerical. Due to many factors affecting the efficiency of isolation and culture of preantral follicles, the improvement of techniques geared to fertility preservation in higher mammals seems to be at an impasse. We need an objective view of the current stand to understand how to progress further. As such, a survey was conducted to analyze the relative distribution of studies performed in ten mammalian species on preantral follicle culture available on PubMed. Using the bovine as a reference model, we explore some factors influencing data variation that contribute to the difficulty in reproducing studies. While years of research have enabled the recapitulation of folliculogenesis from as modest as the early antral follicle stage ex vivo, in vitro preantral folliculogenesis remains elusive. Herein, we revisit the classical evidence that laid the foundations for understanding preantral folliculogenesis and review the length, breadth, and depth of information that the era of big data has currently levied. Moving forward, we recognize the urgency of synthesizing the multi-disciplinary approaches to mimic folliculogenesis in vitro to achieve a translational landscape of infertility at individual and large-scale conservation levels.

In brief:The proposed culture system improves the current state of in vitro culture of growing oocytes in the bovine species and allows access to the untapped gamete reserve, thus improving reproductive efficiency. Abstract:The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus-oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-ethynyl uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes. The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.

Application: Improve the exploitation of the ovarian reserve with the development of a culture system from primordial to secondary follicles.

Acquisition of developmental competence is a complex process in which many cell types cooperate to support oocyte maturation, fertilisation, and preimplantation embryonic development. In recent years, compelling evidence has shown that Progesterone Receptor Membra Component 1 (PGRMC1) is expressed in many cell types of the mammalian reproductive system where it exerts diverse functions. In the ovary, PGRMC1 affects follicular growth by controlling cell viability and proliferation of granulosa cells. PGRMC1 has also a direct role in promoting a proper completion of bovine oocyte maturation, as altering its function leads to defective chromosome segregation and polar body extrusion. Strikingly, the mechanism by which PGRMC1 controls mitotic and meiotic cell division seems to be conserved, involving an association with the spindle apparatus and the chromosomal passenger complex through Aurora kinase B. Conclusive data on a possible role of PGRMC1 in the preimplantation embryo are lacking and further research is needed to test whether the mechanisms that are set in place in mitotic cells also govern blastomere cleavage and subsequent differentiation. Finally, PGRMC1 is also expressed in oviductal cells and, as such, it might also impact fertilisation and early embryonic development, although this issue is completely unexplored. However, the study of PGRMC1 function in the mammalian reproductive system remains a complex matter, due to its pleiotropic function.

In brief RNA granules travel through the cumulus cell network of transzonal projections which is associated with oocyte developmental competence, and RNA packaging involves RNA-binding proteins of the Fragile X protein family. Abstract The determinants of oocyte developmental competence have puzzled scientists for decades. It is known that follicular conditions can nurture the production of a high-quality oocyte, but the underlying mechanisms remain unknown. Somatic cumulus cells most proximal to the oocyte are known to have cellular extensions that reach across the zona pellucida and contact with the oocyte plasma membrane. Herein, it was found that transzonal projections (TZPs) network quality is associated with developmental competence. Knowing that ribonucleoparticles are abundant within TZPs, the distribution of RNA-binding proteins was studied. The Fragile X-related proteins (FXR1P and FXR2P) and two partnering protein families, namely cytoplasmic FMRP-interacting protein and nuclear FMRP-interacting protein, exhibited distinctive patterns consistent with roles in regulating mRNA packaging, transport, and translation. The expression of green fluorescent protein (GFP)–FMRP fusion protein in cumulus cells showed active granule formation and their transport and transfer through filipodia connecting with neighboring cells. Near the projections’ ends was found the cytoskeletal anchoring protein Filamin A and active protein synthesis sites. This study highlights key proteins involved in delivering mRNA to the oocyte. Thus, cumulus cells appear to indeed support the development of high-quality oocytes via the transzonal network.

Oocyte in vitro maturation (IVM) is still a major challenge in human and animal assisted reproduction. Gradual instead of abrupt activation of the ovulatory cascade during IVM has been proposed to enhance nuclear-cytoplasmic synchrony and cumulus-oocyte communication, thus favoring oocyte developmental competence. Herein, we assessed the effects of neuregulin 1 (NRG1), an EGF-like factor that modulates EGFR signaling, on oocyte nuclear maturation dynamics, cumulus expansion and expression of mRNAs regulating these processes during IVM, as well as on post-IVF embryo development following AREG-stimulated IVM in cattle. In experiment 1, cumulus-oocyte complexes (COCs) were subjected to IVM with graded doses of NRG1 (1, 10 or 100 ng/mL) for 6, 9, 12, 20, and 24 h, after which oocyte nuclear status and cumulus mRNA expression were assessed. At 6 h of IVM, NRG1 at 1 ng/mL significantly decreased the percentage of GVBD (germinal vesicle breakdown) oocytes without altering later meiotic dynamics or the percentage of oocytes achieving meiosis II. In experiment 2, adding NRG1 (1 ng/mL) to the IVM medium did not affect cumulus expansion but increased the percentage of expanded and hatched blastocysts, and blastocyst total cell number following IVF/IVC. NRG1 decreased EGFR mRNA abundance while increasing NPR2 and PTX3 mRNA levels at 9 h, and TNFAIP6 mRNA abundance at 20 h of IVM. This is the first study that reports the modulatory effect of NGR1 during oocyte maturation in a mono-ovulatory species and demonstrates that this action may be applied during IVM to improve post-IVF embryo development.

In the past four decades, the bovine model has been highly informative and inspiring to assisted reproductive technologies (ART) in other species. Most of the recent advances in ART have come from studies in cattle, particularly those unveiling the importance of several processes that must be recapitulated in vitro to ensure the proper development of the oocyte. The maintenance of structural and functional communications between the cumulus cells and the oocyte and a well-orchestrated chromatin remodeling with the gradual silencing of transcriptional activity represent essential processes for the progressive acquisition of oocyte developmental competence. These markers are now considered the milestones of physiological approaches to increase the efficiency of reproductive technologies. Different in vitro approaches have been proposed. In particular, the so-called "pre-IVM" or "prematuration" is a culture step performed before in vitro maturation (IVM) to support the completion of the oocyte differentiation process. Although these attempts only partially improved the embryo quality and yield, they currently represent a proof of principle that oocytes retrieved from an ovary or an ovarian batch shouldn't be treated as a whole and that tailored approaches can be developed for culturing competent oocytes in several species, including humans. An advancement in ART's efficiency would be desirable in carnivores, where the success is still limited. Since the progress in reproductive medicine has often come from comparative studies, this review highlights aspects that have been critical in other species and how they may be extended to carnivores.