Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Background: Glycodelin (Gd), a dimeric glycoprotein, appears in the female and male reproductive tract in isoforms like GdA from amniotic fluid and endometrium, GdF from follicular fluid, GdS from seminal plasma and on sperm surface as well. Little is known about the role of Gd in IVF. The aim of our study was to investigate the influence of the Gd-amount in supernatants of IVF-cultures to fertilization success. Methods: Employing monoclonal antibody (mAb) M4f8 soluble Gd levels were evaluated by an enzyme-linked immunosorbent assay (ELISA). We assayed 107 supernatants after 18 h in-vitro fertilisation of single cultured oocytes (S18) with 100000 motile spermatozoa derived from 11 couples undergoing conventional IVF procedure. The results have been compared to blanks comprised of sperm suspensions incubated without any oocytes. The Gd-content in S18 were correlated to fertilization success and characterized by PN-scoring 18 h after insemination. Furthermore we evaluated supernatants (n = 21) of oocytes after ICSI. Total protein (TP) of all supernatants was assessed for calculating the Gd/TP-ratio. Results: The soluble Gd values were calculated for the cultures belonging to each couple. The levels of Gd differed interindividually by a wide range from 4.9 up to 69.2 ng/mL (0.1–3.0% Gd/TP) and in the blanks 2.0–59.5 ng/mL (0.1–4.5% Gd/TP) as well. We associated a couple specific Gd level from 10.0–60.0 ng/mL with a high fertilisation rate (FR = 88%). Both a soluble Gd at a lower level in sperm–oocyte suspensions and a S18 > 60.0 ng/mL were accompanied by a decreased FR (25%). The supernatants (n = 21) after ICSI (FR = 14%) were found Gd-free. Conclusions: Gd-amounts in S18 fluctuated both between the different patients and within their individual sperm–egg cultures. Therefore the protein is suggested not being introduced by spermatozoa only but rather by cumulus cell effects. Beside other parameters one of the fertilisation-dependant factors may be the patient-specific Gd concentration in the surrounding medium.

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PACAP-mediated sperm–cumulus cell interaction promotes fertilization

Reproduction ◽

10.1530/rep-10-0201 ◽

2011 ◽

Vol 141 (2) ◽

pp. 163-171 ◽

Cited By ~ 30

Author(s):

Ichiro Tanii ◽

Tadashi Aradate ◽

Kouhei Matsuda ◽

Akira Komiya ◽

Hideki Fuse

Keyword(s):

Sperm Motility ◽

Acrosome Reaction ◽

Cumulus Cell ◽

Cumulus Cells ◽

Cell Interaction ◽

Fertilization Rate ◽

Type I ◽

Dependent Manner ◽

Sperm Penetration

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus–oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus–oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.

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Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†

Biology of Reproduction ◽

10.1093/biolre/ioaa196 ◽

2020 ◽

Author(s):

Aslihan Turhan ◽

Miguel Tavares Pereira ◽

Gerhard Schuler ◽

Ulrich Bleul ◽

Mariusz P Kowalewski

Keyword(s):

Oocyte Maturation ◽

Cell Function ◽

Hypoxia Inducible Factor ◽

Cumulus Cell ◽

Cumulus Cells ◽

Negative Effects ◽

Protein Levels ◽

Maturation In Vitro

Abstract Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus–oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

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Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations

Zygote ◽

10.1017/s0967199400003798 ◽

1997 ◽

Vol 5 (2) ◽

pp. 117-125 ◽

Cited By ~ 12

Author(s):

Lalantha R. Abeydeera ◽

Hiroaki Funahashi ◽

Nam-Hyung Kim ◽

Billy N. Day

Keyword(s):

Penetration Rate ◽

Calf Serum ◽

Cumulus Cells ◽

In Vitro Fertilisation ◽

Dependent Manner ◽

North Carolina State University ◽

Porcine Oocyte ◽

Low Incidence ◽

Boar Spermatozoa

SummaryPorcine oocyte-cumulus complexes were cultured in bovine serum albumin(BSA)-free North Carolina State University (NCSU)23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml)and hormonal supplements (eCG and hCG: 10IU/ml each) for 22h. They were then cultured in the same medium but without hormonal supplements for an additional 22h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM)199 containing caffeine (5mM), fetal calf serum (FCS; 10%) and varying concentrations (26–56 mM)of NaHCO3 for 9h(experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%)and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p<0.05)at 36–56mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p< 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56mM NaHCO3, respectively. The fertilisation medium containing 46mM NaHCO3 and 1% FCS showed a higher penetration rate (84%)with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

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Apoptosis in bovine cumulus–oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation

Reproduction ◽

10.1530/rep.1.00761 ◽

2005 ◽

Vol 130 (6) ◽

pp. 857-868 ◽

Cited By ~ 29

Author(s):

Paola Pocar ◽

Daniela Nestler ◽

Michaela Risch ◽

Bernd Fischer

Keyword(s):

Cell Apoptosis ◽

Polychlorinated Biphenyl ◽

Cumulus Cell ◽

Cumulus Cells ◽

Coplanar Pcbs ◽

Apoptotic Gene ◽

Commercial Mixture ◽

Bovine Oocytes

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocytein vitromaturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs duringin vitromaturation has been investigated. Results indicate that exposure of cumulus–oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.

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The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

10.26686/wgtn.17006569 ◽

2021 ◽

Author(s):

◽

Zaramasina Lena Clark

Keyword(s):

Gap Junction ◽

Cumulus Cell ◽

Indirect Evidence ◽

Cumulus Cells ◽

Reproductive Technologies ◽

Regulatory Pathway ◽

Meiotic Arrest ◽

Intracellular Cgmp

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study. The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest. Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro. The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>

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Role of Tetraspanin CD9 Molecule in Fertilization of Mammals

Physiological Research ◽

10.33549/physiolres.932876 ◽

2015 ◽

pp. 279-293 ◽

Cited By ~ 9

Author(s):

J. JANKOVIČOVÁ ◽

M. SIMON ◽

J. ANTALÍKOVÁ ◽

P. CUPPEROVÁ ◽

K. MICHALKOVÁ

Keyword(s):

Acrosome Reaction ◽

Vitro Fertilization ◽

Fertilization Process ◽

Sperm Binding ◽

Sperm Penetration ◽

Ambiguous Data ◽

Precise Role ◽

Candidate Molecule

Fertilization process is a very clever and unique process comprising some essential steps resulting in formation of zygote. Tetraspanin CD9 is considered to be a serious candidate molecule participating in these events. The importance of CD9 has been discussed in relation to acrosome reaction, sperm-binding, sperm-penetration, sperm-egg fusion and eventually, egg activation. The abundant expression of CD9 oocyte plasma membrane and the presence of CD9-containing vesicles in the perivitelline space of intact oocytes have been confirmed. Despite the fact that majority of authors analyzed CD9 expressed on oocytes, several studies considered the function of sperm CD9, too. To understand CD9 involvement, various conditions of in vitro fertilization (IVF) assays using polyclonal as well as monoclonal antibodies or knockout mice were carried out. However, ambiguous data have been obtained about the importance of CD9 in sperm-egg binding or fusion. Although the current findings did not prove any hypothesis, the indispensable role of CD9 in fertilization process was not excluded and the precise role of CD9 remains unexplained.

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317 ANTI-HYALURONIDASE ACTION OF ELLAGIC ACID EFFECTIVELY PREVENTS POLYSPERMY THROUGH SUPPRESSION OF THE ACROSOME REACTION INDUCED BY THE SPERM - ZONA INTERACTION DURING PORCINE IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab317 ◽

2007 ◽

Vol 19 (1) ◽

pp. 274

Author(s):

I. Tokeshi ◽

H. Tatemoto ◽

N. Muto ◽

T. Yoshimoto ◽

S. Nakamura ◽

...

Keyword(s):

Ellagic Acid ◽

Acrosome Reaction ◽

Radical Scavenging Activity ◽

Radical Scavenging ◽

Fluorescein Isothiocyanate ◽

Cumulus Cells ◽

Vitro Fertilization ◽

Sperm Binding ◽

Sperm Penetration

We previously reported that the anti-hyaluronidase agents oligosaccharide and tannic acid (TA) were efficient probes for promoting the normal fertilization process in terms of an effective decrease in the incidence of polyspermy, not only in cumulus-enclosed but also in denuded oocytes in pigs. It was unclear, however, why the polyspermic penetration into the zona pellucida (ZP) was directly prevented by the anti-hyaluronidase action. The present study was conducted to examine the effects of 3 tannin relatives [TA, gallic acid (GA), and ellagic acid (EA)] on IVF parameters and the acrosome reaction induced by the sperm–ZP interaction. The anti-hyaluronidase and radical-scavenging activities of tannin relatives were measured by the colorimetric and the DPPH methods, respectively. Porcine cumulus–oocyte complexes (COCs) were cultured for 44 h in 0.1 mL of TCM-199 supplemented with 0.6 mM cysteine, 40 mU mL-1 of FSH, 20 mU mL-1 of LH, and 10% porcine follicular fluid. After in vitro maturation (IVM), the COCs were freed from their cumulus cells and inseminated by frozen-thawed ejaculated sperm in modified Tris-buffered medium (IVF medium) containing 0 (control) or 5 �g mL-1 of tannin relatives. After 2 h of co-incubation, the oocytes were gently pipetted to remove loosely bound sperm and stained with Hoechst 33342 to count the number of ZP-bound sperm, or stained with fluorescein isothiocyanate (FITC)-PNA, PI, and 422,6-diamidino-2-phenylindole to evaluate the acrosomal status. At 10 h post-insemination, IVF parameters were examined by lacmoid staining. The data were analyzed by ANOVA and the Tukey-Kramer test. None of the tannin relatives caused a protective proteolytic modification of the ZP matrix or a reduction of the acrosomal proteolytic activity or the number of ZP-bound sperm. There was no difference in the sperm penetration rate even in the presence of tannin relatives (73-82%). However, the incidence of polyspermy was remarkably prevented by TA (32%; 31/98) and EA (21%; 20/94) compared with the control (58%; 58/100; P < 0.05), resulting from their strong anti-hyaluronidase actions, whereas GA without the anti-hyaluronidase action had no effect on the prevention of polyspermy (51%; 43/84). The rate of acrosome reaction induced by the sperm–ZP interaction was decreased by TA (15%; 123/833) and EA (16%; 110/708) compared with the control (25%; 238/939; P < 0.05), and a similar result was found in sperm binding to the pretreated ZP with 500 U of hyaluronidase for 2 h (18%; 351/1959). Interestingly, when sperm were incubated in IVF medium with 10 �g mL-1 of progesterone for 0.5 h to induce a compulsory acrosome reaction instead of the ZP, EA never disturbed the acrosome reaction (23%; 98/424) as control (23%; 102/437), although this reaction was blocked by TA (13%; 57/427) and GA (13%; 50/375), which possessed higher levels of radical-scavenging activity than EA (P < 0.05). These results indicate that the anti-hyaluronidase action of TA and EA effectively prevented polyspermy during porcine IVF as a consequence of suppression of the acrosome reaction functionally induced by the sperm–ZP interaction requiring the hyaluronidase intervention.

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Tissue plasminogen activator (tPA) of paternal origin is necessary for the success of in vitro but not of in vivo fertilisation in the mouse

Reproduction Fertility and Development ◽

10.1071/rd18175 ◽

2019 ◽

Vol 31 (3) ◽

pp. 433 ◽

Cited By ~ 1

Author(s):

Francisco A. García-Vázquez ◽

C. Soriano-Úbeda ◽

R. Laguna-Barraza ◽

M José Izquierdo-Rico ◽

Felipe A. Navarrete ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Cumulus Cells ◽

Human Reproduction ◽

Sperm Binding ◽

Tissue Plasminogen ◽

Plasmin Inhibitors

Besides its fibrinolytic function, the plasminogen–plasmin (PLG–PLA) system is also involved in fertilisation, where plasminogen activators bind to plasminogen to produce plasmin, which modulates sperm binding to the zona pellucida. However, controversy exists, depending on the species, concerning the role of the different components of the system. This study focused its attention on the role of the PLG–PLA system on fertilisation in the mouse with special attention to tissue plasminogen activator (tPA). The presence of exogenous plasminogen reduced invitro fertilisation (IVF) rates and this decline was attenuated by the presence of plasmin inhibitors in combination with plasminogen. The incubation of spermatozoa with either oocytes or cumulus cells together with plasminogen did not change the acrosome reaction but reduced the number of spermatozoa attached. When spermatozoa from tPA−/− mice were used, the IVF rate decreased drastically, although the addition of exogenous tPA during gamete co-incubation under invitro conditions increased fertilisation success. Moreover, fertility could not be restored after invivo insemination of tPA−/− spermatozoa in the female ampulla, although tPA−/− males were able to fertilise invivo. This study suggests a regulatory role of the PLG–PLA system during fertilisation in the mouse with possible implications in human reproduction clinics, such as failures in tPA production, which could be partially resolved by the addition of exogenous tPA during IVF treatment.

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The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

10.26686/wgtn.17006569.v1 ◽

2021 ◽

Author(s):

◽

Zaramasina Lena Clark

Keyword(s):

Gap Junction ◽

Cumulus Cell ◽

Indirect Evidence ◽

Cumulus Cells ◽

Reproductive Technologies ◽

Regulatory Pathway ◽

Meiotic Arrest ◽

Intracellular Cgmp

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study. The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest. Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro. The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>

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