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The objective of this study was to determine rabbit seminal plasma enzyme activity. Furthermore, correlations between semen parameters and enzyme activity and male age were examined. The study was performed using 17 New Zealand White males from 5 to 9 mo old. Overall, 252 semen samples were collected from bucks from May to September. Semen characteristics were analysed and the seminal plasma was obtained by centrifugation. The activities of alanyl aminopeptidase (APN), aspartate transaminase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALKP) in the seminal plasma fluid were measured. Significant differences between males were found in APN, GGT, LDH, ALKP and ALT activities (P

Neurogenesis in the adult mammalian brain relies on the lifelong persistence of quiescent neural stem cell (NSC) reservoirs. Little is known about the mechanisms that lead to the initial establishment of quiescence, the main hallmark of adult stem cells, during development. Here we show that protein aggregates and autophagy machinery components accumulate in developmental radial glia-like NSCs as they enter quiescence and that pharmacological or genetic blockade of autophagy disrupts quiescence acquisition and maintenance. Conversely, increasing autophagy through AMPK/ULK1 activation instructs the acquisition of the quiescent state without affecting BMP signaling, a gatekeeper of NSC quiescence during adulthood. Selective ablation of Atg7, a critical gene for autophagosome formation, in radial glia-like NSCs at early and late postnatal stages compromises the initial acquisition and maintenance of quiescence during the formation of the hippocampal dentate gyrus NSC niche. Therefore, we demonstrate that autophagy is cell-intrinsically required to establish NSC quiescence during hippocampal development. Our results uncover an important role of autophagy in the transition of developmental NSCs into their dormant adult form, paving the way for studies directed at further understanding the mechanisms of stem cell niche formation and maintenance in the mammalian brain., (© 2023. The Author(s).)

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical β-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active β-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo . Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 García-Corzo, Calatayud-Baselga, Casares-Crespo, Mora-Martínez, Julián Escribano-Saiz, Hortigüela, Asenjo-Martínez, Jordán-Pla, Ercoli, Flames, López-Alonso, Vilar and Mira.)

The present study was conducted to characterise rabbit sperm proteins focusing on the influence of the genetic origin. Six samples were recovered during two months from five males from genotype A (New Zealand White origin) and five from genotype R (California origin). Sperm proteins were extracted and subjected to in-gel digestion nano LC-MS/MS and bioinformatics analysis. The resulting library included 487 identified proteins validated with ≥95% Confidence (unused Score ≥ 1.3). All the identified proteins belonged to Oryctolagus cuniculus taxonomy. These data are available via ProteomeXchange with identifier PXD007989. Only 7 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Regarding the comparison of the sperm proteins abundance between genotypes, forty proteins were differentially expressed. Among them, 25 proteins were over-expressed in genotype A, while 15 proteins were over-expressed in genotype R. In conclusion, this study characterizes for the first time rabbit sperm proteins and provides evidence that genotype is related to a specific abundance of spermatozoa proteins., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adult neurogenesis persists in the adult mammalian brain due to the existence of neural stem cell (NSC) reservoirs in defined niches, where they give rise to new neurons throughout life. Recent research has begun to address the implication of constitutive (basal) autophagy in the regulation of neurogenesis in the mature brain. This review summarizes the current knowledge on the role of autophagy-related genes in modulating adult NSCs, progenitor cells and their differentiation into neurons. The general function of autophagy in neurogenesis in several areas of the embryonic forebrain is also revisited. During development, basal autophagy regulates Wnt and Notch signaling and is mainly required for adequate neuronal differentiation. The available data in the adult indicate that the autophagy-lysosomal pathway regulates adult NSC maintenance, the activation of quiescent NSCs, the survival of the newly born neurons and the timing of their maturation. Future research is warranted to validate the results of these pioneering studies, refine the molecular mechanisms underlying the regulation of NSCs and newborn neurons by autophagy throughout the life-span of mammals and provide significance to the autophagic process in adult neurogenesis-dependent behavioral tasks, in physiological and pathological conditions. These lines of research may have important consequences for our understanding of stem cell dysfunction and neurogenic decline during healthy aging and neurodegeneration.

The present study was designed to prove new rabbit insemination extenders containing aminopeptidase inhibitors (AMIs) with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. In addition, different hormone concentrations were tested in these extenders, evaluating their in vivo effect on rabbit reproductive performance after artificial insemination. A total of 911 females were inseminated with semen diluted with the four experimental extenders (C4 group: 4 μg buserelin/doe in control medium (Tris-citric acid-glucose supplemented with bestatin 10 μM and EDTA 20 mM), C5 group: 5 μg of buserelin/doe in control medium, Q4 group: 4 μg of buserelin/doe into CS-DS nanoparticles in control medium, Q5 group: 5 μg of busereline/doe into CS-DS nanoparticles in control medium). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 versus 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied (P > 0.05). We conclude that the CS-DS nanoparticles prepared by a coacervation process as carrier for buserelin acetate allows reducing the concentration of hormone used in extenders supplemented with bestatin and EDTA without affecting the fertility and prolificacy of rabbit females., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study was conducted to characterise rabbit seminal plasma proteins (SP proteins) focusing on the influence of the genetic origin and seasonality. In addition, β-NGF protein quantity in SP was determined. Semen samples were recovered from January to December 2014 using 6 males belonging to genotype A and six from genotype R. For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment. A total of 24 pools (3 for each season and genetic line) were analysed. SP proteins of the two experimental groups were recovered and subjected to in-solution digestion nano LC-MS/MS and bioinformatics analysis. The resulting library included 402 identified proteins validated with ≥95% Confidence (unused Score ≥ 1.3). These data are available via ProteomeXchange with identifier PXD006308. Only 6 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Twenty-three proteins were differentially expressed between genotypes, 11 over-expressed in genotype A and 12 in genotype R. Regarding the effect of season on rabbit SP proteome, results showed that there is no clear pattern of protein variation throughout the year. Similar β-NGF relative quantity was observed between seasons and genotypes. In conclusion, this study generates the largest library of SP proteins reported to date in rabbits and provides evidence that genotype is related to a specific abundance of SP proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, β nerve growth factor (β-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as β-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and β-NGF, they could be used to develop new extenders with less hormone concentration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study was designed to develop chitosan (CS)-dextran sulphate (DS) nanoparticles containing a GnRH analogue and to study their effect on rabbit (Oryctolagus cuniculus) semen quality. Six experimental extenders were tested as follows: (control) Tris-citric acid-glucose (TCG), (1) 0.05% CS-0.05% DS (4:1), (2) 0.1% CS-0.05% DS (4:1), (3) 0.05% CS-0.05% DS (3:1), (4) 0.1% CS-0.05% DS (3:1), (5) 0.1% CS-0.05% DS (2:1). CS and DS were dissolved in TCG medium, and nanoparticles were obtained through magnetic stirring. Rabbit seminal samples were incubated up to 5 hr at 37°C in the extenders, and seminal quality was evaluated. The entrapment efficiency was 40%-50%. After 5 hr at 37°C, a 20% of the hormone was released. Results showed that the presence of CS-DS nanoparticles did not affect rabbit semen motility, viability and membrane functionality; however, acrosome integrity was significantly higher versus control (p < .001)., (© 2017 Blackwell Verlag GmbH.)

The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI-TOF/TOF or LC-MS/MS analysis and were the following ones: FAM115E-like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E-like (113 kDa), haemoglobin subunit zetalike (13 kDa) and β-nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies., (© 2016 Blackwell Verlag GmbH.)

The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of aminopeptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 ± 0.26 and 9.3 ± 0.23 vs. 8.2 ± 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size., (Copyright © 2016 Elsevier Inc. All rights reserved.)

One of the greatest challenges for reproductive cryobiologists today is to develop an efficient cryopreservation method for human and domestic animal oocytes. The objective of the present study was to optimize a low toxicity solution called VM3 to vitrify porcine oocytes using an open pulled straw (OPS) device and to evaluate the effects on viability, chromosomal organization and cortical granules distribution. Two experiments were conducted in this study. Firstly, we determined the minimum concentration of cryoprotectant present in the VM3 solution required (7.6 M) for vitrification using an OPS device. The appearance of opacity was observed when using a cooling solution at -196°C; no observable opacity was noted as vitrification. In addition, the ultrastructure of oocytes in VM3 or VM3 optimized solution was examined using cryo-scanning electron microscopy. The minimum total cryoprotectant concentration present in VM3 solution necessary for apparent vitrification was 5.6 M when combined with use of an OPS device. Use of both vitrification solutions showed a characteristic plasticized surface. In the second experiment, the relative cytotoxicity of vitrification solutions (VM3 and VM3 optimized) was studied. Oocyte viability, chromosomal organization and the cortical granules distribution were assessed by fluorescent stain. After warming, oocyte survival rate was similar to that of fresh oocytes. The vitrification process significantly reduced correct chromosomal organization and cortical granules distribution rates compared with the fresh oocytes group. However, correct chromosomal organization and cortical granules distribution rates did not differ among oocytes placed in different vitrification solutions. In conclusion, our data demonstrated that the VM3 solution can be optimized and that reduction in concentration to 5.6 M enabled vitrification of oocytes with an OPS device, however use of the VM3 optimised solution had no beneficial effect on vitrification of porcine oocytes.

The study tested the efficacy of pre-treating mature porcine oocytes with cytochalasin B before vitrification by the open pulled straw method (OPS) in a low toxicity solution containing ice blockers. The effects of pre-treating the oocytes with 7.5 micrograms per ml cytochalasin B before vitrification on membrane integrity, chromosome organisation and cortical granule distribution were evaluated. When oocytes pre-treated with cytochalasin B before vitrification were compared with control oocytes, similar membrane integrity was observed. In contrast, when both vitrified oocytes groups (treated and untreated with cytochalasin B) were compared with fresh oocytes, significantly lower proportions of oocytes with normal chromosomes aligned regularly on the metaphase plate and peripheral cortical granule distribution were observed. The percentages of oocytes with normal chromosomes aligned regularly on the metaphase plate were similar between those treated or untreated with cytochalasin B before vitrification. Similar results were found for normal cortical granules distribution. Irrespective of previous cytochalasin B exposure, vitrification gave rise to higher abnormal cortical granule distribution percentages. Cytochalasin B pretreatment of oocytes before vitrification does not help to reduce the damage induced by the cryopreservation process of porcine oocytes.