Reproduction Fertility and Development (REPROD FERT DEVELOP )

Publisher: Commonwealth Scientific and Industrial Research Organization (Australia); Fertility Society of Australia; Australian Academy of Science; Australian Society for Reproductive Biology; Society for Reproductive Biology, CSIRO Publishing

Description

Reproduction, Fertility and Development is an international journal for the publication of original and significant contributions related to reproduction and developmental biology in humans, domestic animals and wildlife. Contributions may take the form of research articles, reviews, short communications or viewpoint articles that deal with the scientific aspects of reproductive and developmental physiology, biochemistry, endocrinology, immunology, cell biology, genetics and behaviour, and the applications of reproductive technologies in humans, livestock, wildlife and pest management.

Impact factor 2.58

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    Impact factor
  • 5-year impact
    2.56
  • Cited half-life
    6.80
  • Immediacy index
    0.49
  • Eigenfactor
    0.01
  • Article influence
    0.66
  • Website
    Reproduction, Fertility and Development website
  • Other titles
    Reproduction fertility and development
  • ISSN
    1031-3613
  • OCLC
    19505713
  • Material type
    Conference publication, Periodical, Internet resource
  • Document type
    Journal / Magazine / Newspaper, Internet Resource

Publisher details

CSIRO Publishing

  • Pre-print
    • Author can archive a pre-print version
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    • Author can archive a post-print version
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    • On author's personal repository or institutional repository
    • Must link to publisher version
    • Published source must be acknowledged
    • Publisher's version/PDF cannot be used
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48h and 144h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (PPPP<0.05) of cleavage and blastocyst formation by 48 and 144h after IVF compared with all other groups. These results indicate that supplementing with 0.005mM glucuronic acid and 0.005mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) antidepressant commonly prescribed during pregnancy and lactation. Pre- and post-partum depression, as well as SSRI treatment during these periods, may change maternal care, interfering with offspring development. Moreover, it is known that SSRIs may alter testes structure and function in offspring. The present study investigated the effects of maternal FLX exposure on maternal behaviour and testes function in offspring. Female Wistar rats were treated with 7.5mgkg-1 FLX or tap water (control group) by gavage from the Day 1 of pregnancy until 21 days after birth (postnatal Day (PND) 21). Maternal behaviour was evaluated and morphofunctional analyses of offspring testes were conducted on PND 21 and 50. There were no significant differences between the FLX-treated and control groups regarding maternal behaviour. Nor did maternal treatment with FLX have any effect on bodyweight gain, anogenital distance, day of preputial separation, testis weight and the gonadosomatic index in male offspring. However, there was a decreased number of Sertoli cells at both PND 21 and 50 in FLX-exposed male offspring. The findings of the present study demonstrate that maternal exposure to FLX can impair testicular function in weanling and pubertal animals.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: Overall efficiency of in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition, and supplementation. Brain-derived neurotrophic factor (BDNF), which is a physiologically important neurotrophin, has been used to enhance oocyte maturation in some previous studies (Lee et al. 2007; Zhang et al. 2010). However, the efficacy of BDNF to improve oocyte competence has not been fully established especially in ovine. Therefore, the present study aimed to evaluate the effect of BDNF during in vitro maturation (IVM) on maturation rate, intracellular glutathione (GSH) content, and embryonic development in sheep oocytes. Cumulus-oocyte complexes (COC) were obtained from ovaries of ewes. The COC were placed in maturation medium supplemented with either 10 (IVM-B10) or 100 (IVM-B100) ngmL(-1) of BDNF (PeproTech, London, UK). Oocytes in control group were incubated in the same maturation medium without BDNF. The IVM was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24h. After IVM, several oocytes from the IVM-B10 (n=110), IVM-B100 (n=124), and control (n=110) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. To measure GSH content, several oocytes from the IVM-B10 (n=28), IVM-B100 (n=33), and control (n=37) groups were incubated in tyrodes medium containing 10µM Cell Tracker blue for 30min and transferred under fluorescence microscope, with digital images analysed by image J software. To evaluate the embryonic development, several oocytes from IVM-B10 (n=145), IVM-B100 (n=137), and control (n=143) groups were subjected to parthenogenetic activation by applying 1min of exposure to 2.5µM ionomycin followed by 2mM 6-DMAP treatment for 3h. After stimulation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated previously. Four replications were performed. The metaphase-II rate, cleavage, and blastocyst rates were compared by x(2) analysis. The GSH content was analysed by one-way ANOVA. A P-value of less than 0.05 was considered significant. The results showed that metaphase-II rate was higher in the IVM-B100 group (88.7%), as compared with the control group (77.3%), but not significant as compared with that in the IVM-B10 group (84.5%). No difference was also found between the IVM-B10 group and control group in terms of the metaphase-II rate. Oocytes in the IVM-B10 group revealed a higher (96.8%) GSH content than both of the IVM-B100 (86.9%) and control (86.3%) groups. There was, however, no difference in the GSH content between the IVM-B100 group and control group. The proportion of cleaved embryos was not different between the groups; however, the blastocyst rate was higher in both the IVM-B10 (37.9%) and IVM-B100 (39.3%) groups compared with the control group (22.4%). Collectively, the results of this study showed that supplementation of IVM media with BDNF promoted nuclear maturation, increased GSH content, and stimulated in vitro embryonic development in ovine.
    41st Annual Conference of the IETS, Versailles, France; 01/2015
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    ABSTRACT: It is a general belief that as soon as the oocyte is recovered from the follicular environment, the nuclear maturation starts spontaneously in vitro, while specific stimulation for the cytoplasmic maturation is lacking (Gilchrist and Thompson 2007 Theriogenology 67, 6-15; Albuz et al. 2010 Hum. Reprod. 25, 2999-3011). As both nuclear and cytoplasmic maturation are required to prepare the oocyte for successful fertilization and embryonic development, a defective cytoplasmic maturation might be an important cause of low blastocyst rates in vitro (Albuz et al. 2010 Hum. Reprod. 25, 2999-3011). Nuclear and cytoplasmic maturation can be evaluated using fluorescent dyes. Assessment of nuclear maturation is typically based on the visualisation of chromatin, whereas cytoplasmic maturation is evaluated by the localization of cytoplasmic organelles [i.e. the cortical granules (CG)]. Equine oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation (IVM; Smits et al. 2010 Vlaams Diergen. Tijds. 79, 134-138), oocytes were fixed and permeabilized. Subsequently, CG were labelled by incubation in 10µgmL(-1) FITC-labelled lens culinaris agglutinin during 15min at RT. Chromatin was counterstained to verify the nuclear status with 20µgmL(-1) Hoechst 33342 during 15min at RT. Stained oocytes with no or dispersed chromatin were classified as degenerated. Based on the absence or presence of the first polar body (PB), non-degenerated oocytes were either classified as nuclear immature (MI, no PB present) or nuclear mature (MII, PB present). The non-degenerated oocytes were further subdivided in 3 categories based on the migration of the CG: 1) cytoplasmic immature oocytes with (clusters of) CG randomly distributed throughout the ooplasm, 2) oocytes in transition stage with progressing CG migration to the oocyte cortex, and 3) cytoplasmic mature oocytes with a clearly visible CG monolayer just underneath the oolemma. The mean and standard deviation of nuclear and cytoplasmic parameters were calculated using Excel (Excel 2007, Microsoft Corp., Redmond, WA, USA). In 3 replicates, 86.6±2.75% of all oocytes (131/151) demonstrated a corresponding nuclear and cytoplasmic maturation pattern (MI corresponding to CG1 and 2; MII corresponding to CG3). Only 12.0±2.82% of the oocytes (16/133) revealed a cytoplasmic maturation pattern (CG 1 or 2) that lagged behind the nuclear maturation (MII). On the other hand, 22.2±9.8% of the oocytes (4/18) were already cytoplasmic (CG3) but not yet nuclear matured (MI). Since most of the equine in vitro matured oocytes exhibited, surprisingly, a corresponding nuclear and cytoplasmic maturation pattern, it can be concluded that the gap between the nuclear and cytoplasmic maturation in vitro is less important than is generally believed. Consequently, the IVM step is not the main obstacle to increase the efficiency of the in vitro production process in horses.
    41st Annual IETS conference - Reproduction, fertility, and development, Versailles, France; 01/2015
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    ABSTRACT: A variety of controlled mild stressors have been applied for activation of temporary response in oocytes, embryos, and somatic cells. So far, several stressors have been used to induce mild stress, including that of hydrostatic pressure, osmotic stress, mechanical stress, and oxidative challenges. Based on these evidences, we hypothesised that the ethanol in sublethal concentration would be capable of generating mild stress that may ultimately leads to an adaptive response in spermatozoa. To evaluate this hypothesis, semen samples (n=24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled for each replicate. Pooled samples were divided into 5 equal parts and each part diluted with tris-glycerol-based (Optidyl(®)) extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9), and 0.15 (O-E15) % (vol/vol) ethanol and frozen. After thawing, sperm motility and velocity parameters (sperm class analysis), apoptosis status (Phospatidylserin Translocation Detection commercial kit), plasma membrane integrity (eosin-Nigrosin staining), malondialdehyde concentration (thiobarbituric acid reaction), and mitochondrial activity (rhodamine-123 and propidium iodide) were evaluated. The data were analysed using Proc Mixed of SAS 9.1 (version 9.1; SAS Institute Inc., 2002, Cary, NC, USA). Tukey's test was used to compare least squares means. As a result, the O-E9 group showed higher (85.2%) percentage of total motility compared with O-E0 (73.6%), O-E3 (51.9%), and O-E15 (67.5%) groups (P<0.05). A highest (P<0.05) percentage of live spermatozoa were observed in the O-E9 (62.9%) group as compared with O-E0 (49.4%), O-E3 (50.3%), and O-E15 (49.6%) groups, and also the proportion of apoptotic spermatozoa in the O-E9 (10.6%) group tended to be lowest as compared with those of O-E0 (15.6%), O-E3 (17.2%), O-E15 (14.1%) groups (P>0.05). The plasma membrane integrity was higher (P<0.05) in O-E9 (90.8%) compared with O-E3 (75%) and O-E15 (77.2%) groups; however, the difference was not significant when the O-E9 group was compared with the O-E0 group (83.2%; P>0.05). Obtained results revealed that malondialdehyde level was lower in O-E3 (1.03%), O-E9 (0.63%), and O-E15 (0.89%) groups compared with the O-E0 (1.94%) group (P<0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in O-E9 (57.7%) and O-E15 (57.5%) groups compared with O-E0 (49.1%) and O-E3 (38.2%) groups (P<0.05). These results strongly suggest that supplementation of Optidyl(®) extender with sublethal concentration of ethanol influences post-thawed bull sperm quality in a dose-dependent manner. However, further studies are needed to empirically determine the effect of supplementation on fertilization and pregnancy outcome.
    41st Annual Conference of the IETS, Versailles, France; 01/2015
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    ABSTRACT: The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: Endometrial epithelium plays a crucial role in the first immune response to invading bacteria by producing cytokines and chemokines. The aim of this study was to investigate the first inflammatory response of the endometrium in vivo and in vitro. Gene expression of several pro-inflammatory factors and Toll-like receptors (TLR2, -4, -6) was determined in endometrial cytobrush samples obtained from healthy cows and cows with clinical or subclinical endometritis. Endometrial epithelial cells were co-cultured with an isolated autochthonous uterine bacterial strain Bacillus pumilus. Total RNA was extracted from in vivo and in vitro samples and subjected to real-time reverse transcription polymerase chain reaction. CXC ligands (CXCL) 1/2 and CXC chemokine receptor (CXCR) 2 mRNA expression was higher in cows with subclinical endometritis and CXCL3 mRNA expression was higher in cows with clinical endometritis compared with healthy cows. B. pumilus induced cell death of epithelial cells within 24h of co-culturing. The presence of B. pumilus resulted in significantly higher mRNA expression of interleukin 1? (IL1A), IL6, IL8, CXCL1-3 and prostaglandin-endoperoxide synthase 2 in co-cultured cells compared with untreated controls. The maximum increase was mainly detected after 2h. These results support the hypothesis that bacterial infection of endometrial cells might induce prompt synthesis of pro-inflammatory cytokines resulting in a local inflammatory reaction.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: The expression and activity of matrix metalloproteinases (MMPs) may be regulated by oxidative stress in various pathophysiological processes; therefore, the aim of the present study was to analyse the associations between the expression of the gelatinases MMP-9 and MMP-2 and their tissue inhibitors TIMP-1, TIMP-2 and levels of total antioxidant capacity (TAC) and advanced oxidation protein products (AOPP) in seminal plasma prepared for artificial insemination. Levels of MMPs and TIMPs were evaluated using ELISA, whereas TAC and AOPP in the seminal plasma of 131 childless men and 38 fertile volunteers were determined spectrophotometrically. Seminal MMP-9 expression was higher in childless men than in fertile subjects, whereas there was no significant differences in MMP-2 expression between the analysed seminal groups. TIMP-1 and TIMP-2 expression was similar in all groups. However, TAC expression was significantly higher in infertile normozoospermic and oligozoospermic men and AOPP expression was higher in astheno-, oligo- and normozoospermic infertile patients than in fertile men. High AOPP, together with an increased MMP-9:TIMP-1 ratio alters the oxidative-antioxidative balance of the ejaculate, thereby reducing male fertility, and therefore these parameters may serve as additional diagnostic markers of semen quality and male reproductive potential.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: Transcription factor AP-2? (TFAP2C) is a member of the transcription factor activating enhancer binding protein (AP) family. In the present study we determined the temporal and spatial expression patterns of TFAP2C in porcine parthenotes during preimplantation development. Porcine TFAP2C transcripts were expressed at all stages of preimplantation development, with highest expression at the 8-cell stage. In contrast with the mouse, TFAP2C protein was not restricted to the trophectoderm and was also detected in the ICM in blastocyst stage porcine parthenotes. In knockdown (KD) experiments, most TFAP2C-depleted embryos were arrested before the compacted 8-cell stage. This developmental failure is attributed to abnormal expression of genes involved in cell adhesion, tight junction biogenesis and cell proliferation. Interestingly, although the conserved region 4 (CR4) of the porcine OCT4 5? upstream regionlacked the AP2C-binding motif, OCT4 transcript levels were elevated in porcine TFAP2C-KD 8-cell embryos, suggesting TFAP2C may be involved in the regulation of OCT4 in porcine embryos through other mechanisms. In summary, the results suggest that TFAP2C is necessary for the transition from de novo transcript synthesis by activation to compaction and further development, and the different expression patterns of TFAP2C in porcine embryos may reflect species-specific functions during preimplantation embryo development.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: This study was designed to determine whether prenatal dexamethasone treatment has an effect on follicular development and atresia in the ovary of spiny mouse (Acomys cahirinus) offspring. Dexamethasone (125µg kg-1 bodyweight per day) was administered to pregnant spiny mice from Day 20 of gestation to parturition. The processes of follicle loss were analysed using classical markers of apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling reaction, active caspase-3) and autophagy (Lamp1). The present study indicated that dexamethasone reduced the pool of healthy primordial follicles. Moreover, the oocytes from these follicles showed intensive caspase-3 and Lamp1 staining. Surprisingly, dexamethasone caused an increase in the number of secondary follicles; however, most of these follicles were characterised by extensive degeneration of the oocyte and caspase-3 and Lamp1 labelling. Western-blot analysis indicated that the glucocorticoid receptor as well as apoptosis and autophagy markers were more strongly expressed in the DEX-treated group than in the control. On the basis of these findings, we have concluded that dexamethasone impairs spiny mouse folliculogenesis and enhances follicular atresia through induction of autophagy or combined autophagy and apoptosis.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: We investigated the local modulation of some histochemical properties of oviducts of the dromedary (Camelus dromedarius), focusing on the immnolocalisation of hyaluronic acid (HA) synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL1) and the HA receptor CD44 in the ampulla and isthmus. Abundant acidic mucopolysaccharides (glycosaminoglycans) were detected by Alcian blue staining along the luminal surface of both ciliated and non-ciliated epithelial cells (LE). Staining for HAS2 was higher in the primary epithelial folds of the ampulla compared with the isthmus, especially in secretory cells, adluminal epithelial surface and supranuclear cell domain. HAS3 staining was stronger in the LE of the isthmus than ampulla. HYAL2 was detected in the LE in the ampulla and isthmus and was more intense in the adluminal projections of secretory cells. HYAL1 was weakly detected in the LE with no difference between the ampulla and isthmus. Strong CD44 immunostaining was present in the LE of the ampulla and isthmus. CD44 staining was higher in secretory cells than in ciliated epithelial cells and was higher in the supranuclear region than the basal region of the cytoplasm. In conclusion, we provide evidence that HA synthesis and turnover occur in the camel oviduct. Differences in HAS2 and HAS3 expression suggest regional differences in the molecular size of HA secreted in oviductal fluid that may influence oviduct-gamete interaction in the camel.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: Use of the dietary supplement quercetin is on the rise. Because previous studies imply an inhibitory effect of quercetin on male fertility, we explored the effects of this flavonoid on fertility in female mice. Birth outcomes, and ovarian morphology in 4-week-old offspring, were assessed in mice receiving dietary quercetin (5mgkg-1day-1) for 9 months during two breeding periods: from 2 to 6 months (prime reproductive age) and 8 to11 months of age. Quercetin increased birth spacing, leading to a 60% reduction in the number of litters, but enhanced folliculogenesis in ovaries of female offspring. While in young females quercetin caused an almost 70% increase in litter size, in older animals this effect was reversed. Consistent with the inhibitory activity of quercetin on the enzyme transglutaminase 2 (TG2), genetic ablation of TG2 in mice mirrors the effects of quercetin on birth outcomes and follicular development. Further, TG2-null mice lack responsiveness to quercetin ingestion. Our study shows for the first time that dietary quercetin can cause reduced reproductive potential in female mice and implies that TG2 may regulate ovarian ageing.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: The oocyte-derived growth factor bone morphogenetic protein (BMP) 15 plays important roles in fertility, but its mechanism of action differs between species. Generation of BMP15-binding molecules, as an essential investigation tool, would be helpful to provide valuable insight into the underlying biological features of BMP15. The BMP15-binding molecules could be antibodies or aptamers. Aptamers have many advantages over antibodies as macromolecular ligands for target proteins. DNA aptamers can be obtained by a method of Systematic Evolution of Ligands by EXponential enrichment (SELEX) beginning with a pool of random sequences. However, the success of this technique cannot be guaranteed if the initial pool lacks candidate sequences. Herein, we report on the creation of DNA aptamers by means of modified SELEX. The modification included enhanced mutation and progressive selection during an in vitro evolutionary process. As a proof-of-principle, we started from a single sequence instead of a multiple-sequence pool. Functional aptamers against the recombinant BMP15 were successfully created and identified.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: During early pregnancy the endometrium undergoes a major transformation in order for it to become receptive to blastocyst implantation. The actin cytoskeleton and plasma membrane of luminal uterine epithelial cells (UECs) and the underlying stromal cells undergo dramatic remodelling to facilitate these changes. Filamin A (FLNA), a protein that crosslinks actin filaments and also mediates the anchorage of membrane proteins to the actin cytoskeleton, was investigated in the rat uterus at fertilisation (Day 1) and implantation (Day 6) to determine the role of FLNA in actin cytoskeletal remodelling of UECs and decidua during early pregnancy. Localisation of FLNA in UECs at the time of fertilisation was cytoplasmic, whilst at implantation it was distributed apically; its localisation is under the influence of progesterone. FLNA was also concentrated to the first two to three stromal cell layers at the time of fertilisation and shifted to the primary decidualisation zone at the time of implantation. This shift in localisation was found to be dependent on the decidualisation reaction. Protein abundance of the FLNA 280-kDa monomer and calpain-cleaved fragment (240kDa) did not change during early pregnancy in UECs. Since major actin cytoskeletal remodelling occurs during early pregnancy in UECs and in decidual cells, the changing localisation of FLNA suggests that it may be an important regulator of cytoskeletal remodelling of these cells to allow uterine receptivity and decidualisation necessary for implantation in the rat.
    Reproduction Fertility and Development 01/2015;
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    ABSTRACT: We tested whether the reversible effects of nutrition on spermatogenesis in sexually mature sheep were mediated by Sertoli cells. Rams were fed with diets designed to achieve a 10% increase (High), no change (Maintenance) or a 10% decrease (Low) in body mass after 65 days. At the end of treatment, testes were lighter in the Low than the High group (P<0.01). The Maintenance group had intermediate values that were not significantly different from those of the other two groups. Spermatogenesis (Johnsen score) was impaired in the Low group, but normal in both other groups. There was no effect of treatment on Sertoli cell numbers, although 1% of Sertoli cells appeared to retain their ability to proliferate. By contrast, Sertoli cell function was affected by dietary treatment, as evidenced by differences between the High and Low groups (P<0.05) in the expression of seven Sertoli cell-specific genes. Under-nutrition appeared to reverse cellular differentiation leading to disruption of tight-junction morphology. In conclusion, in sexually mature sheep, reversible reductions in testis mass and spermatogenesis caused by under-nutrition were associated with impairment of basic aspects of Sertoli cell function but not with changes in the number of Sertoli cells.
    Reproduction Fertility and Development 01/2015; Published online.
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    ABSTRACT: Granulosa cells (GC) are important constituents of the follicular environment for oocyte competence acquisition. However, their functionality depends on oocyte-derived factors, such as GDF9 and BMP15, which act through BMPRII receptor signalling. Gene silencing using lipofection has been used as an important tool to investigate the role of cell genes and proteins. The aim of this study was to establish the ideal conditions for lipofection in bovine GC and starting from this protocol to establish a methodology for silencing of the BMPRII gene by RNA interference, and to use this strategy to study the functions of BMPRII in GDF9 signalling. GC were obtained from slaughterhouse ovaries by aspiration of follicles (2 to 6mm) and subsequently cultured in DMEM medium at 38.5°C and 5% CO2 in air. All data analyzes were performed by GraphPad Prism(®) version 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using one-way ANOVA followed by Tukey's test. For optimizing the conditions for lipofection, the GC were treated with different amounts of lipofection agents Lipofectamine(®) RNAiMAX (Invitrogen, Carlsbad, CA, USA; 1, 2, and 3µL) or Lipofectamine(TM) 2000 (Invitrogen; 1, 2, and 3µL) and the transfection indicators Siglo(®) (GE Healthcare, Waukesha, WI, USA; 30 to 100nM) or FUGW transgenic plasmid (100 to 900nM) during 24 and 48h. The highest efficiency of lipofection was observed at 24h of culture with 2μL of Lipofectamine™ 2000+100nM of Siglo(®). Based on these conditions, different concentrations of siBMPRII (100 to 500pM) were tested during 24h of culture and subsequently, different incubation times (0, 6, 12, 18, and 24h) with the best siRNA concentration in order to establish optimum conditions for gene silencing. GC were evaluated for the relative abundance of mRNA for BMPRII using PPIA and β-actin as endogenous controls by real-time PCR. All concentrations provided similar and highly significant transcript reduction in comparison to control (P<0.001), so the lowest of them was adopted (100pM). For different incubation times with 100pM siBMPRII also a similar decrease was also seen, which was more significant at 24h (P<0.01). The best concentration (100pM) and incubation time (24h) with the siRNA were analysed by Western blotting, which confirmed the BMPRII reduction also at protein level (P<0.05). For functional evaluation, GC submitted or no to gene silencing were incubated with or without GDF9 (100ng) and assessed for expression of genes controlled by GDF9 through its BMPRII receptor (LHR, INHA, and INHBA), besides the CYP17 gene, a marker of potential theca cells contamination. LHR expression was reduced in silenced groups (P<0.05) and highly suppressed by GDF9 in non-silenced groups (P<0.001), while INHA and INHBA expression remained constant in the different groups (P>0.05), suggesting that the control of these genes in bovine does not behave as reported in other species. The lack of amplification CYP17 indicates the nonexistence of the contamination. In conclusion, this study allowed the establishment of an efficient methodology for the silencing of BMPRII by lipofection in bovine GC, which may be used as a tool for the functional study of BMPRII and potentially for other genes of interest.
    41st IETS Annual Conference, Versailles, France; 01/2015
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    ABSTRACT: The aim of the present study was to investigate the hypothesis that parental periconception nutrition in adult seahorses affects the development and growth of their offspring. We tested the hypothesis that because seahorse embryos develop inside the male's brood pouch, manipulation of the male's diet would affect offspring growth and development independently of the female's diet. Adult males and females were fed separately with either wild-caught crustaceans or commercial aquarium diet for 1 month before conception to influence the periconception environment. Approximately 10000 offspring were obtained from four different treatment groups (Male/Wild or Male/Commercial×Female/Wild or Female/Commercial). Weights, physical dimensions and fatty acid profiles of the newborns were determined. Offspring produced when the males receiving commercial diet were mated with wild-fed females were larger (P<0.05) than those produced by wild-fed males. When both males and females were fed with commercial diet, their offspring were significantly smaller than those from the other treatment groups. When commercial diet-fed females were mated with wild-fed males, the offspring showed distortion of the snout:head length ratio. These results support the view that the preconception diet received by males and females differentially affects embryonic development.
    Reproduction Fertility and Development 12/2014;