Naproxen administration affects murine late folliculogenesis, reduces granulosa cell proliferation and the number of ovulated oocytes.

Naproxen reduces the production of prostaglandins via inhibition of the cyclooxygenase. Studies have shown that its administration in women can be related to failed ovulation. Therefore, preclinical investigations must be performed in order to investigate its effects in experimental models. Thus, the aim of this study was to evaluate the effects of naproxen on murine folliculogenesis, ovulation, and female fertility. Female C57BL/6 mice (n = 128 - 6 weeks old) were divided into Control, low (10 mg/kg), and high naproxen (50 mg/kg) groups, who were treated for 8 days and directed to morphofunctional analyses. Follicular quantification showed a reduced percentage of antral follicles in naproxen-treated animals. These treated animals also showed smaller oocytes included in secondary and antral follicles, and the diameter of secondary and antral follicles was also reduced. A reduction in the percentage of Ki67-positive granulosa cells was observed in treated animals that also showed down-regulation of Igf1r compared to control. After an ovarian stimulation protocol, naproxen-treated animals showed a reduction in the percentage of secondary and antral follicles, a reduced number of ovulated oocytes and, corpora lutea, and an increased number of failed ovulations. Finally, naproxen-treated animals also showed a reduction in mating index and pregnancy rate. Our findings suggested that, in mice, naproxen administration (eight days treatment) negatively affects molecular and morphological aspects related to late folliculogenesis, ovulation, and fertility.

Acute Exposure to Two Biocides Causes Morphological and Molecular Changes in the Gill Ciliary Epithelium of the Invasive Golden Mussel Limnoperna fortunei (Dunker, 1857).

Limnoperna fortunei, the golden mussel, is a bivalve mollusk considered an invader in South America. This species is responsible for ecological and economic damages due to its voluminous fouling capability. Chemical biocides such as MXD-100™ and sodium dichloroisocyanurate (NaDCC) are often used to control L. fortunei infestations in hydraulic systems. Thus, we proposed to investigate the effects of different periods (24, 48 and 72 h) of exposure to MXD-100™ (0.56 mg L-1) and NaDCC (1.5 mg L-1) on the gills of L. fortunei through morphological and molecular analyses. NaDCC promoted progressive morphological changes during the analyzed periods and only an upregulation of SOD and HSP70 expression during the first 24 h of exposure. MXD-100™ led to severe morphological changes from the first period of exposure, in addition to an upregulation of SOD, CAT, HSP70 and CYP expression during the first 24 h. In contrast, MXD-100™ led to a downregulation of CAT transcription between 24 and 48 h. In static conditions, NaDCC causes lethal damage after 72 h of exposure, and that exposure needs to be continuous to achieve the control of the species. Meanwhile, the MXD-100™ treatment presented several effects during the first 24 h, showing acute toxicity in a shorter period of time.

Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212 nm vs. 284 ± 130 nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Drastic Loss of Antral Follicles Due to Gene Expression Dysregulation Occurs on the First Day After Subcutaneous Ovarian Transplantation.

Ovarian cryopreservation is an alternative for the preservation of fertility, and the subcutaneous transplantation site is considered one of the most promising. Studies evaluating the follicular growth and its relationship with gene expression and vascular perfusion are essential for improving this technique and its clinical application. Thus, the aim of this study was to evaluate the effect of subcutaneous autotransplantation and vitrification on follicular growth and atresia and their relationship with vascular perfusion and gene expression. Therefore, female mice were ovariectomized, and the ovaries were divided in two experimental groups (1) vitrified (treatment, n = 97) and (2) not vitrified (control, n = 97) and subsequently were transplanted. Then grafts, from both groups, were recovered after 1, 12, or 23 days (D1, D12, D23) and subjected to follicular quantification, morphometry, and qPCR. Non-transplanted ovaries (D0) were also used. The estrous cycle and vascular perfusion were monitored throughout the experiment. On D9, 100% of the animals had reestablished their estrous cycles (p > 0.05). Blood perfusion at the transplant site was similar for both treatments (p > 0.05), with greater perfusion at the site of vitrified transplants only on D1 (p < 0.05). A drastic reduction in the number of antral follicles and an increased number of atretic follicles were observed on D1 (p < 0.0001), associated with upregulation of Casp3, Fshr, and Igf1r; and downregulation of Bax, Acvr1, Egfr, and Lhcgr (p < 0.05). Our findings indicate that the first day after subcutaneous transplantation is a critical period for follicular survival, with intense follicular atresia independent of Bax upregulation.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

RGMa can induce skeletal muscle cell hyperplasia via association with neogenin signalling pathway.

Although originally discovered inducing important biological functions in the nervous system, repulsive guidance molecule a (RGMa) has now been identified as a player in many other processes and diseases, including in myogenesis. RGMa is known to be expressed in skeletal muscle cells, from somites to the adult. Functional in vitro studies have revealed that RGMa overexpression could promote skeletal muscle cell hypertrophy and hyperplasia, as higher efficiency in cell fusion was observed. Here, we extend the potential role of RGMa during C2C12 cell differentiation in vitro. Our results showed that RGMa administrated as a recombinant protein during late stages of C2C12 myogenic differentiation could induce myoblast cell fusion and the downregulation of different myogenic markers, while its administration at early stages induced the expression of myogenic markers with no detectable morphological effects. We also found that RGMa effects on skeletal muscle hyperplasia are performed via neogenin receptor, possibly as part of a complex with other proteins. Additionally, we observed that RGMa-neogenin is not playing a role as an inhibitor of the BMP signalling in skeletal muscle cells. This work contributes to placing RGMa as a component of the mechanisms that determine skeletal cell fusion via neogenin receptor.

Time course and side-by-side analysis of mesodermal, pre-myogenic, myogenic and differentiated cell markers in the chicken model for skeletal muscle formation.

The chicken is a well-established model for amniote (including human) skeletal muscle formation because the developmental anatomy of chicken skeletal muscle matches that of mammals. The accessibility of the chicken in the egg as well as the sequencing of its genome and novel molecular techniques have raised the profile of this model. Over the years, a number of regulatory and marker genes have been identified that are suited to monitor the progress of skeletal myogenesis both in wildtype and in experimental embryos. However, in the various studies, differing markers at different stages of development have been used. Moreover, contradictory results on the hierarchy of regulatory factors are now emerging, and clearly, factors need to be able to cooperate. Thus, a reference paper describing in detail and side-by-side the time course of marker gene expression during avian myogenesis is needed. We comparatively analysed onset and expression patterns of the key markers for the chicken immature paraxial mesoderm, for muscle-competent cells, for cells committed to myogenesis and for cells entering terminal differentiation. We performed this analysis from stages when the first paraxial mesoderm is being laid down to the stage when mesoderm formation comes to a conclusion. Our data show that, although the sequence of marker gene expression is the same at the various stages of development, the timing of the expression onset is quite different. Moreover, marker gene expression in myogenic cells being deployed from the dorsomedial and ventrolateral lips of the dermomyotome is different from those being deployed from the rostrocaudal lips, suggesting different molecular programs. Furthermore, expression of Myosin Heavy Chain genes is overlapping but different along the length of a myotube. Finally, Mef2c is the most likely partner of Mrf proteins, and, in contrast to the mouse and more alike frog and zebrafish fish, chicken Mrf4 is co-expressed with MyoG as cells enter terminal differentiation.

The emergence of Pax7-expressing muscle stem cells during vertebrate head muscle development.

Pax7 expressing muscle stem cells accompany all skeletal muscles in the body and in healthy individuals, efficiently repair muscle after injury. Currently, the in vitro manipulation and culture of these cells is still in its infancy, yet muscle stem cells may be the most promising route toward the therapy of muscle diseases such as muscular dystrophies. It is often overlooked that muscular dystrophies affect head and body skeletal muscle differently. Moreover, these muscles develop differently. Specifically, head muscle and its stem cells develop from the non-somitic head mesoderm which also has cardiac competence. To which extent head muscle stem cells retain properties of the early head mesoderm and might even be able to switch between a skeletal muscle and cardiac fate is not known. This is due to the fact that the timing and mechanisms underlying head muscle stem cell development are still obscure. Consequently, it is not clear at which time point one should compare the properties of head mesodermal cells and head muscle stem cells. To shed light on this, we traced the emergence of head muscle stem cells in the key vertebrate models for myogenesis, chicken, mouse, frog and zebrafish, using Pax7 as key marker. Our study reveals a common theme of head muscle stem cell development that is quite different from the trunk. Unlike trunk muscle stem cells, head muscle stem cells do not have a previous history of Pax7 expression, instead Pax7 expression emerges de-novo. The cells develop late, and well after the head mesoderm has committed to myogenesis. We propose that this unique mechanism of muscle stem cell development is a legacy of the evolutionary history of the chordate head mesoderm.