Werner Giehl Glanzner

The regulation of transcriptional activity constitutes a primary layer of control over cellular function across diverse tissues and cell types. This regulatory transcriptional landscape is largely governed by epigenetic mechanisms, including DNA and histone modifications, the dynamic activity of epigenetic “writers” and “erasers,” and the influence of small regulatory molecules such as microRNAs (miRNAs). miRNAs are small non-coding RNA molecules that play critical roles in transcriptional and post-transcriptional gene regulation. By modulating the expression of transcription factors, kinases, and other regulatory proteins, miRNAs exert tight control over cellular processes and maintain homeostasis. Given the essential requirement for precise cellular coordination in reproductive biology, miRNAs have emerged as key regulators of reproductive function. They participate in multiple stages of reproductive processes, ranging from ovarian function to the establishment and maintenance of pregnancy, thereby influencing fertility outcomes across species. In this review, we summarize recent advances in understanding the roles of miRNAs in key reproductive events, including gametogenesis, oocyte maturation, embryonic genome activation, early embryonic development, implantation and pregnancy. We aim to highlight the principal miRNAs involved in the maintenance of reproductive function and fertility, and their molecular targets and the regulatory networks when known. Based on our review, we conclude that although their potential in clinical and/or agricultural applications still remains uncertain, their role in regulating fundamental aspects of cellular biology, particularly reproductive processes, is becoming increasingly clear.

While embryo transfer (ET) has not yet been widely adopted in the commercial swine industry, it is an essential tool in biomedical research, including the creation of gene‐edited pigs. Traditionally, ET in pigs is performed by open surgery (laparotomy), an effective but invasive and time‐consuming method associated with risks such as infection and herniation. To overcome these limitations, we developed a minimally invasive laparoscopic embryo transfer (LapET) method enabling ET into the oviduct using a sheep insemination device (Aspic, IMV Technologies, France). Seventeen gilts were synchronised with 17.6 mg altrenogest daily for 16 days, 10 mg dinoprost on D15, 1250 IU eCG on D16 and 1000 IU hCG on D19, followed by LapET on D21. Each gilt received an average of 160 embryos, transferred 24–48 h post‐somatic cell nuclear transfer (SCNT). Under general anaesthesia, gilts were placed on a surgical table in a 30° incline Trendelenburg position. Three trocars were inserted to serve as ports for the laparoscope, a grasper and the Aspic pipette. The grasper served to expose the ovaries to confirm ovulation and to position the oviduct for embryo deposition. Following ET, the oviduct was released, instruments were removed and incisions were closed with surgical glue. Pregnancy was confirmed by ultrasonography, with 12 of 17 gilts (70.6%) diagnosed as pregnant, a success rate comparable to our recent laparotomy‐based SCNT ETs into the oviduct (71.4%; 10 of 14). These results demonstrate that LapET is a viable, fast, minimally invasive and effective alternative to traditional open surgery ET in swine.

Marmosets are a valuable model for studying human disorders, but developing specific genetic models requires efficient protocols for embryo genomic manipulation. Although marmoset embryos have been produced in vitro, oocyte retrieval traditionally involves extended recombinant human follicle-stimulating hormone (FSH) treatment (9–10 days), surgical laparotomy and ovarian exposure, limiting repeat procedures. Here we evaluated a simpler, cost-effective ovarian stimulation protocol using porcine pituitary FSH, combined with laparoscopic ovum pick-up (LOPU), for in vitro production of marmoset embryos via in vitro fertilization (IVF) and somatic cell nuclear transfer. A total of 3,922 oocytes were retrieved from 129 LOPU procedures, averaging 30.4 oocytes per LOPU, with 85.5% (26.0 per LOPU) graded as viable. The procedure was safe, with no adverse effects observed in ovaries or fimbria after up to nine LOPU sessions. Most collected oocytes were meiotically immature and matured in vitro before use. Following IVF, approximately 40% of oocytes cleaved, and 40% of cleaved embryos developed to the expanded blastocyst stage. LOPU-derived oocytes also supported somatic cell nuclear transfer, with 69.8% of embryos cleaving and 21.8% forming blastocysts. IVF-derived blastocyst quality was assessed by total cell count, immunodetection of SOX2 (inner cell mass) and CDX2 (trophectoderm), and cryotolerance. Embryo transfer to recipients resulted in successful live births. These findings demonstrate that a simplified pituitary FSH protocol followed by LOPU is an efficient, less invasive and safe method for retrieving developmentally competent marmoset oocytes, offering a promising approach for advancing marmoset-based research in disease modeling and reproductive biotechnology.

Embryo genome activation (EGA) is a crucial event implicated in proper embryonic development. Similarly, the DNA Damage Response (DDR) is essential for correcting or preventing occasional errors during embryonic cell division that could result in embryo development arrest or the propagation of genetic abnormalities. Although both EGA and DDR are regulated by epigenetic mechanisms, the role of microRNAs (miRNAs) in these processes has not been explored in pig embryos. Herein, we assessed the abundance of miRNAs linked to embryo development and DDR across four developmental stages: Day 2 (D2), Day 3 (D3), and Day 4 (D4), and at the blastocyst stage, as well as after DNA damage induction. mRNA levels of EGA‐related genes confirmed our timepoints as representing EGA timeframe, while immunofluorescence for γH2AX validated DNA damage induction. Significant decrease in blastocyst rate and total cell number per blastocyst was detected in UV‐exposed embryos. Analysis of miRNA abundance revealed increased levels of miR‐200a‐5p on D3, which were partially maintained on D4. Both miR‐15a and miR‐24‐3p increased on D4, but their levels were downregulated in UV‐exposed embryos at the same stage of development. In blastocysts, UV exposure upregulated miR‐29a‐3p and miR‐344b‐3p. Together, these findings provide the first characterization of miRNAs expression in porcine embryos during EGA and following DNA damage induction.

Purpose Somatic cell nuclear transfer (SCNT) is a valuable tool for investigating reprogramming mechanisms and creating animal clones for applications in production, conservation, companionship, and biomedical research. However, SCNT efficiency remains low. Expression of nuclear proteins associated with an undifferentiated chromatin state, such as the oocyte-specific variant of the linker histone H1 (H1foo), represents a strategy for improving reprogramming outcomes, but this approach has not been tested in the context of SCNT. Methods Bovine H1foo (bH1foo) was transfected into porcine fibroblasts via electroporation for expression until SCNT. The transcriptomic profile of these cells was analyzed, and their potential as donor cells for SCNT was evaluated 48 h post-electroporation. Results Strong nuclear localization of bH1foo persisted for 48 h post-electroporation. A total of 447 genes were differentially expressed, and lower levels of H3K4me3 and H3K27me3 were detected in bH1foo-expressing cells, indicating changes in chromatin remodeling and function. Embryo development and total cell number per blastocyst were similar between SCNT embryos produced with control and bH1foo-expressing cells. mRNA levels of genes involved in embryonic genome activation were comparable between embryos derived from control and bH1foo-expressing cells on days 3 and 4 of development, suggesting that bH1foo did not disrupt this critical process. Conclusions The heterologous expression of bovine H1foo altered the chromatin function of porcine fibroblasts without impairing development to the blastocyst stage following SCNT. These results highlight the potential of expressing nuclear proteins as a strategy to enhance cell reprogramming and cloning efficiency, including interspecies cloning applications.

The nuclear factor erythroid 2–related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions.

: In brief: Ubiquitination plays a pivotal role in a multitude of cellular functions; however, the precise contributions of various ubiquitin ligases in governing early developmental processes remain largely unexplored. This study revealed that the E3 ubiquitin ligases DCAF13 and RNF114 are both necessary for the normal regulation of early porcine embryo development. Ubiquitylation is required for normal regulation of many biological functions by modulating several protein facets such as structure, stability, interaction, localization, and degradation. In this study, we explored the roles of two E3 ubiquitin ligases (E3s), the DDB1- and CUL4-associated factor 13 (DCAF13) and the Ring finger protein 114 (RNF114), in the regulation of porcine embryo development. Attenuation of DCAF13 mRNA decreased embryo development at the blastocyst stage, while the development of RNF114-attenuated embryos was not significantly different than that of control embryos. The average number of cells per blastocyst was decreased in DCAF13-attenuated embryos and increased in RNF114-attenuated embryos compared to controls. The relative mRNA abundance of the histone methyltransferase SUV39H1, which regulates histone H3 lysine 9 trimethylation (H3K9me3), was increased in both DCAF13- and RNF114-attenuated embryos, but nuclear immunofluorescence signal for H3K9me3 on day 3 embryos was not significantly altered between attenuated and control embryos. Nuclear immunofluorescence signal for H3K4m3 was decreased in DCAF13-attenuated embryos, but it was increased in RNF114-attenuated embryos compared to controls. Attenuation of DCAF13 and RNF114 mRNAs increased transcript levels for the DNA recombinase RAD51 and decreased expression of phosphorylated histone H2A.X (γH2AX), which suggests an impact on DNA damage repair. In addition, lower mRNA expression of the lysine demethylases 5B (KDM5B) and 5C (KDM5C), both involved in embryo genome activation and DNA repair, was detected in DCAF13-attenuated embryos. These findings indicated that both DCAF13 and RNF114 have important roles in the regulation of the early development of porcine embryos.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects.