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- Borgbo, T, et al.
(författare)
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Effect of the FSH receptor single nucleotide polymorphisms (FSHR 307/680) on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles.
- 2015
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Ingår i: Molecular Human Reproduction. - : Oxford University Press (OUP). - 1460-2407. ; 21:3, s. 255-261
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Tidskriftsartikel (refereegranskat)abstract
- The most pronounced effects of FSH signaling are potentially displayed in follicle fluid (FF), which acts as a reservoir for FSH induced granulosa cell (GC) secreted hormones. This study investigates the effects of two common polymorphisms of FSHR, FSHR 307 (rs6165) and FSHR 680 (rs6166), by evaluating the hormone and gene expression profiles of human small antral follicles collected under physiological conditions in connection with fertility preservation. In total 69 women at various time during the menstrual cycle was included in this study. The intrafollicular hormone content of 179 FF and the gene expression levels of 85 GC samples were correlated to the genotype of both FSHR polymorphisms. The following parameters were evaluated: follicle diameter, levels of Anti-Müllerian Hormone (AMH), progesterone, oestradiol, testosterone and androstenedione, and gene expression levels of FSHR, luteinizing hormone receptor (LHR), androgen receptor (AR), aromatase cytochrome p450 (CYP19A1,) AMH and AMH receptor II (AMHR2). There was 100 % concordance between the FSHR 307 and the FSHR 680 genotypes: A/A (p.307Thr/Thr and p.680Asn/Asn), A/G (p.307Thr/Ala and p.680Asn/Ser), and G/G (p.307Ala/Ala and p.680Ser/Ser). Considering all follicles, compared to the other genotypes the G/G genotype was associated with significantly elevated gene expression levels for LHR, while AMHR2 gene expression levels were significantly reduced. In follicles 3-6 mm in diameter LHR gene expression was significantly increased, whereas AMH gene expression was significantly reduced for the G/G genotype. In follicles>6 mm, oestradiol and CYP19A1 gene expression levels were significantly higher for the G/G genotype. In conclusion, significant changes were observed between the FSHR 307/680 polymorphisms in human small antral follicles collected under physiological FSH conditions.
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| 4. |
- Johannisson, Anders, et al.
(författare)
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Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines
- 2017
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Ingår i: Molecular Human Reproduction. - : Oxford University Press (OUP). - 1360-9947 .- 1460-2407. ; 23, s. 521-534
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Tidskriftsartikel (refereegranskat)abstract
- STUDY QUESTION: Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation?SUMMARY ANSWER: Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling.WHAT IS KNOWN ALREADY: In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process.STUDY DESIGN, SIZE, DURATION: Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 x 10(6) SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5 degrees C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 mu M), Go6983 (PKC inhibitor, 10 mu M), PD98059 (ERK-1/2 inhibitor, 30 mu M), H89 and KT (PKA inhibitors, 50 mu M and 100 nM, respectively), KH7 (sAC inhibitor, 10 mu M), BAPTA-AM (intracellular Ca2+ chelator, 50 mu M), EGTA (10 mu M) and Probenecid (MRPs general inhibitor, 500 mu M). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 mu g/ml) and bicarbonate (40 mM).PARTICIPANTS/MATERIALS, SETTING, METHODS: Straws of frozen bovine semen (20-25 x 10(6) spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried.MAIN RESULTS AND THE ROLE OF CHANCE: In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001).LARGE SCALE DATA: None.LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data.WIDER IMPLICATIONS OF THE FINDINGS: These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies.
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