It was hypothesized the lower fertility of repeat-breeder (RB) Holstein cows is associated with oocyte quality and this negative effect is enhanced during summer heat stress (HS). During the summer ...and the winter, heifers (H; n=36 and 34, respectively), peak-lactation (PL; n=37 and 32, respectively), and RB (n=36 and 31, respectively) Holstein cows were subjected to ovum retrieval to assess oocyte recovery, in vitro embryonic developmental rates, and blastocyst quality terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and total cell number. The environmental temperature and humidity, respiration rate, and cutaneous and rectal temperatures were recorded in both seasons. The summer HS increased the respiration rate and the rectal temperature of PL and RB cows, and increased the cutaneous temperature and lowered the in vitro embryo production of Holstein cows and heifers. Although cleavage rate was similar among groups H=51.7%±4.5 (n=375), PL=37.9%±5.1 (n=390), RB=41.9%±4.5 (n=666), blastocyst rate was compromised by HS, especially in RB cows H=30.3%±4.8 (n=244) vs. 23.3%±6.4 (n=150), PL=22.0%±4.7 (n=191) vs. 14.6%±7.6 (n=103), RB=22.5%±5.4 (n=413) vs. 7.9%±4.3 (n=177). Moreover, the fragmentation rate of RB blastocysts was enhanced during the summer, compared with winter 4.9%±0.7 (n=14) vs. 2.2%±0.2 (n=78) and other groups H=2.5%±0.7 (n=13), and PL=2.7%±0.6 (n=14) suggesting that the association of RB fertility problems and summer HS may potentially impair oocyte quality. Our findings provide evidence of a greater sensitivity of RB oocytes to summer HS.
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The ovarian follicle components must provide an ideal environment to ensure the success of reproductive processes, and communication between follicular cells is crucial to support proper ...oocyte growth. Recently, it has been demonstrated that the presence of extracellular vesicles (EVs) carrying microRNAs (miRNAs) in follicular fluid represents an important autocrine and paracrine communication mechanism inside the ovarian follicle. In this study, we tested the hypothesis that the miRNA content of EVs isolated from ovarian follicular (granulosa and cumulus–oocyte complexes) cell‐conditioned culture media is dependent upon cell type. We initially screened bovine granulosa cells (GCs) and cumulus–oocyte complexes (COCs), as well as their derived EVs for 348 miRNAs using real‐time PCR, and detected 326 miRNAs in GCs and COCs cells and 62 miRNAs in GCs and COCs EVs. A bioinformatics analysis of the identified cell‐specific and differentially expressed miRNAs predicted that they likely modulate important cellular processes, including signalling pathways such as the PI3K‐Akt, MAPK and Wnt pathways. By investigating the origins of miRNAs within the follicular fluid, the results of this study provide novel insights into follicular miRNA content and intercellular communication that may be of invaluable use in the context of reproductive technologies, diagnostic of ovarian‐related diseases and/or the identification of biomarkers for oocyte and embryo quality.
Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the ...embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3′ terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development.
The endometrium is fundamentally required for successful pregnancy in ruminants and species where the posthatching conceptus undergoes a protracted elongation and peri-implantation phase of ...pregnancy. Moreover, there are substantial waves of pregnancy loss during this pre- and peri-implantation period of pregnancy the precise source of which has not been clearly defined i.e., the maternal uterine contribution to this loss. Understanding the molecular interactions required for successful pregnancy in cattle will allow us to intervene to support pregnancy success during this vulnerable window. The endometrium contributes to most key developmental milestones of pregnancy establishment, including (1) contributing to the regulation of the oestrus cycle, (2) nourishing the preimplantation conceptus, (3) responding to the conceptus to create a more receptive microenvironment, (4) providing essential biophysical support, and (5) signalling and producing factors which affect the mother systemically. This review will summarise what we currently know about conceptus-maternal interactions as well as identify the gaps in our knowledge that could be filled with newer in vitro model approaches. These include the use of microfluidics, organ-on-a-chip devices, and bioinformatic approaches. This will help maximise food production efficiency (both meat and dairy) and decrease the environmental burden, while enhancing our understanding of the fundamental processes required for successful implantation in cattle.
Abstract Mammalian fetal survival and growth are dependent on a well-established and functional placenta. Although transient, the placenta is the first organ to be formed during pregnancy and is ...responsible for important functions during development, such as the control of metabolism and fetal nutrition, gas and metabolite exchange, and endocrine control. Epigenetic marks and gene expression patterns in early development play an essential role in embryo and fetal development. Specifically, the epigenetic phenomenon known as genomic imprinting, represented by the non-equivalence of the paternal and maternal genome, may be one of the most important regulatory pathways involved in the development and function of the placenta in eutherian mammals. A lack of pattern or an imprecise pattern of genomic imprinting can lead to either embryonic losses or a disruption in fetal and placental development. Genetically modified animals present a powerful approach for revealing the interplay between gene expression and placental function in vivo and allow a single gene disruption to be analyzed, particularly focusing on its role in placenta function. In this paper, we review the recent transgenic strategies that have been successfully created in order to provide a better understanding of the epigenetic patterns of the placenta, with a special focus on imprinted genes. We summarize a number of phenotypes derived from the genetic manipulation of imprinted genes and other epigenetic modulators in an attempt to demonstrate that gene-targeting studies have contributed considerably to the knowledge of placentation and conceptus development.
There is a molecular crosstalk between the trophoblast and maternal immune cells of bovine endometrium. The uterine cells are able to secrete cytokine/chemokines to either induce a suppressive ...environment for establishment of the pregnancy or to recruit immune cells to the endometrium to fight infections. Despite morphological differences between women and cows, mechanisms for immune tolerance during pregnancy seem to be conserved. Mechanisms for uterine immunesuppression in the cow include: reduced expression of major histocompatability proteins by the trophoblast; recruitment of macrophages to the pregnant endometrium; and modulation of immune‐related genes in response to the presence of the conceptus. Recently, an eGFP transgenic cloned embryo model developed by our group showed that there is modulation of foetal proteins expressed at the site of syncytium formation, suggesting that foetal cell can regulate not only by the secretion of specific factors such as interferon‐tau, but also by regulating their own protein expression to avoid excessive maternal recognition by the local immune system. Furthermore, foetal DNA can be detected in the maternal circulation; this may reflect the occurrence of an invasion of trophoblast cells and/or their fragment beyond the uterine basement membrane in the cow. In fact, the newly description of exosome release by the trophoblast cell suggests that could be a new fashion of maternal‐foetal communication at the placental barrier. Additionally, recent global transcriptome studies on bovine endometrium suggested that the immune system is aware, from an immunological point of view, of the presence of the foetus in the cow during early pregnancy.
Cervical cancer is one of the most common causes of cancer-related deaths in women worldwide. Despite advances in current therapies, women with advanced or recurrent disease present poor prognosis. ...Photodynamic therapy (PDT) has emerged as an effective therapeutic alternative to treat oncological diseases such as cervical cancer. Phthalocyanines (Pcs) are considered good photosensitizers (PS) for PDT, although most of them present high levels of aggregation and are lipophilic. Despite many investigations and encouraging results, Pcs have not been approved as PS for PDT of invasive cervical cancer yet. This review presents an overview on the pathophysiology of cervical cancer and summarizes the most recent developments on the physicochemical properties of Pcs and biological results obtained both in vitro in tumor-bearing mice and in clinical tests reported in the last five years. Current evidence indicates that Pcs have potential as pharmaceutical agents for anti-cervical cancer therapy. The authors firmly believe that Pc-based formulations could emerge as a privileged scaffold for the establishment of lead compounds for PDT against different types of cervical cancer.
SARS-CoV-2 is an enveloped non-segmented positive-sense RNA virus, classified as a beta coronavirus, responsible for the COVID-19 pandemic. Angiotensin-converting enzyme 2 (ACE2), reported as a ...SARS-CoV-2 receptor, is expressed in different human tissues (lung, intestine, and kidney) and in the testis, ovaries, uterus, and vagina. This suggests a potential risk to the human reproductive tract in COVID-19 patients. In addition, SARS-CoV-2 RNA has been detected in the blood, urine, facial/anal swabs, semen, and vaginal secretion, suggesting other potential means of transmission. However, little has been reported about SARS-CoV-2 infection in the male and nonpregnant female reproductive tracts, which may provide direct evidence on sexual transmission and fertility problems. Therefore, we focused this narrative review mainly on the distribution of ACE2 and SARS-CoV-2 positivity in the male and nonpregnant female reproductive tracts, providing an overview of the potential threat of COVID-19 to reproductive health and sexual transmission.
Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the ‘large offspring syndrome’ (LOS) in ruminants. It has been ...hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental‐specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental‐specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.
Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. ...The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.
Overview of the steps for obtaining the iPS cells. From the somatic cell chosen, genic factors are added, and after the formation of colonies, they must be characterized as positive for alkaline phosphatase, embryoid body formation, gene markers for immunofluorescence and real-time PCR, in vivo tumor formation and the possible abnormalities in chromosomes, resulting from reprogramming process. Display omitted
•iPSCs cells were generated by canine fetal fibroblast.•IPSCs were generated without LIF and resistant to enzymatic passages.•The strains were positive for all characterization testes and capable to forming tumor mass after 120 days.•The evaluated strains showed no chromosomal changes.
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