Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

AbstractSoluble adenylyl cyclase (sAC: ADCY10) is essential for activating dormant sperm. Studies of freshly dissected mouse sperm identified sAC as needed for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in human sperm. Unlike dissected mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. Even in ejaculated human sperm, TDI-10229 interrupts stimulated motility and capacitation, and it prevents acrosome reaction in capacitated sperm. At present, there are no non-hormonal, pharmacological methods for contraception. Because sAC activity is required post-ejaculation at multiple points during the sperm’s journey to fertilize the oocyte, sAC inhibitors define candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in females.

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Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaab054 ◽

2021 ◽

Author(s):

Melanie Balbach ◽

Lubna Ghanem ◽

Thomas Rossetti ◽

Navpreet Kaur ◽

Carla Ritagliati ◽

...

Keyword(s):

Adenylyl Cyclase ◽

Ex Vivo ◽

Beat Frequency ◽

Human Sperm ◽

Soluble Adenylyl Cyclase ◽

Mouse Sperm ◽

Acrosomal Exocytosis ◽

Induced Changes ◽

First Time ◽

Sperm Functions

Abstract Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency, and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.

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139. THE REGULATION OF TRP53 IN SPERM

Reproduction Fertility and Development ◽

10.1071/srb09abs139 ◽

2009 ◽

Vol 21 (9) ◽

pp. 58

Author(s):

H. Mudaliar ◽

C. O'Neill

Keyword(s):

Protein Synthesis ◽

Western Blot ◽

Marked Reduction ◽

Acrosome Reaction ◽

Human Sperm ◽

Cell Stress ◽

Time Dependent ◽

Mouse Sperm ◽

Tumour Suppressor Protein ◽

Acute Increase

TRP53 is a tumour suppressor protein that is a universal sensor of cell stress. Upon ejaculation, sperm undergo a process of capacitation which allows them to become fertile. We have previously shown that mouse and human sperm possess TRP53. In this study we analyzed the regulation TRP53 presence in sperm. Mouse sperm were collected from the epididymides and incubated for various times in fertilisation medium. TRP53 was detected by both Western blot analysis and immunolocalization. We found that sperm collected directly from the epididymis generally had little or no detectable TRP53. The level increased with time in culture over a period of 120 min. Most of the TRP53 was detected in the midpiece of sperm although it was also detected in the head of a small proportion of sperm. The increase in TRP53 with time accompanied the increase in the proportion of sperm undergoing the acrosome reaction. Yet, Trp53-null sperm still underwent the acrosome reaction at a normal rate. By contrast, sperm that were prevented from undergoing capacitation and the acrosome reaction (by the removal of albumin or calcium from media) showed a marked reduction in the amount of TRP53 detected. This shows that TRP53 may be dependent upon capacitation, but the reverse was not the case. Inhibition of protein synthesis by puromycin did not block the time-dependent increase in TRP53 in sperm. Canonically, the TRP53 level is controlled by its rate of degradation by MDM2-ubiquitin mediated proteolysis. We found that MDM2 was present in sperm and inhibition of MDM2 (by Nutlin-3) caused an acute increase in TRP53 detected in sperm. This study shows that TRP53 levels are acutely regulated in sperm during the time that sperm acquire the capacity to fertilise, yet sperm lacking TRP53 are capable of fertility. Identification of the role for this TRP53 in sperm is under investigation.

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Low physiological levels of prostaglandins E2 and F2α improve human sperm functions

Reproduction Fertility and Development ◽

10.1071/rd14035 ◽

2016 ◽

Vol 28 (4) ◽

pp. 434 ◽

Cited By ~ 4

Author(s):

Mariana Rios ◽

Daniela V. Carreño ◽

Carolina Oses ◽

Nelson Barrera ◽

Bredford Kerr ◽

...

Keyword(s):

Intracellular Calcium ◽

Sperm Motility ◽

Acrosome Reaction ◽

Seminal Fluid ◽

Human Sperm ◽

Human Spermatozoa ◽

Control Solution ◽

Prostaglandins E2 And F2α ◽

Motility Parameters ◽

Sperm Functions

Prostaglandins (PGs) have been reported to be present in the seminal fluid and cervical mucus, affecting different stages of sperm maturation from spermatogenesis to the acrosome reaction. This study assessed the effects of low physiological PGE2 and PGF2α concentrations on human sperm motility and on the ability of the spermatozoa to bind to the zona pellucida (ZP). Human spermatozoa were isolated from seminal samples with normal concentration and motility parameters and incubated with 1 μM PGE2, 1 μM PGF2α or control solution to determine sperm motility and the ability to bind to human ZP. The effects of both PGs on intracellular calcium levels were determined. Incubation for 2 or 18 h with PGE2 or PGF2α resulted in a significant (P < 0.05) increase in the percentage of spermatozoa with progressive motility. In contrast with PGF2α, PGE2 alone induced an increase in sperm intracellular calcium levels; however, the percentage of sperm bound to the human ZP was doubled for both PGs. These results indicate that incubation of human spermatozoa with low physiological levels of PGE2 or PGF2α increases sperm functions and could improve conditions for assisted reproduction protocols.

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Conserved Mechanism of Bicarbonate-Induced Sensitization of CatSper Channels in Human and Mouse Sperm

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.733653 ◽

2021 ◽

Vol 9 ◽

Author(s):

Juan J. Ferreira ◽

Pascale Lybaert ◽

Lis C. Puga-Molina ◽

Celia M. Santi

Keyword(s):

Human Sperm ◽

Membrane Depolarization ◽

Concentration Increase ◽

Epididymal Sperm ◽

Soluble Adenylyl Cyclase ◽

Mouse Sperm ◽

Intracellular Ca ◽

Sperm Membrane ◽

Human And Mouse ◽

Kinase A

To fertilize an egg, mammalian sperm must undergo capacitation in the female genital tract. A key contributor to capacitation is the calcium (Ca2+) channel CatSper, which is activated by membrane depolarization and intracellular alkalinization. In mouse epididymal sperm, membrane depolarization by exposure to high KCl triggers Ca2+ entry through CatSper only in alkaline conditions (pH 8.6) or after in vitro incubation with bicarbonate (HCO3–) and bovine serum albumin (capacitating conditions). However, in ejaculated human sperm, membrane depolarization triggers Ca2+ entry through CatSper in non-capacitating conditions and at lower pH (< pH 7.4) than is required in mouse sperm. Here, we aimed to determine the mechanism(s) by which CatSper is activated in mouse and human sperm. We exposed ejaculated mouse and human sperm to high KCl to depolarize the membrane and found that intracellular Ca2+ concentration increased at pH 7.4 in sperm from both species. Conversely, intracellular Ca2+ concentration did not increase under these conditions in mouse epididymal or human epididymal sperm. Furthermore, pre-incubation with HCO3– triggered an intracellular Ca2+ concentration increase in response to KCl in human epididymal sperm. Treatment with protein kinase A (PKA) inhibitors during exposure to HCO3– inhibited Ca2+ concentration increases in mouse epididymal sperm and in both mouse and human ejaculated sperm. Finally, we show that soluble adenylyl cyclase and increased intracellular pH are required for the intracellular Ca2+ concentration increase in both human and mouse sperm. In summary, our results suggest that a conserved mechanism of activation of CatSper channels is present in both human and mouse sperm. In this mechanism, HCO3– in semen activates the soluble adenylyl cyclase/protein kinase A pathway, which leads to increased intracellular pH and sensitizes CatSper channels to respond to membrane depolarization to allow Ca2+ influx. This indirect mechanism of CatSper sensitization might be an early event capacitation that occurs as soon as the sperm contact the semen.

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Optimization of lead compounds into on-demand, nonhormonal contraceptives: leveraging a public–private drug discovery institute collaboration†

Biology of Reproduction ◽

10.1093/biolre/ioaa052 ◽

2020 ◽

Vol 103 (2) ◽

pp. 176-182 ◽

Cited By ~ 1

Author(s):

Melanie Balbach ◽

Makoto Fushimi ◽

David J Huggins ◽

Clemens Steegborn ◽

Peter T Meinke ◽

...

Keyword(s):

Drug Discovery ◽

Adenylyl Cyclase ◽

Small Molecule ◽

Male Fertility ◽

Small Molecule Inhibitors ◽

Soluble Adenylyl Cyclase ◽

Lead Compounds ◽

The Third ◽

On Demand

Abstract Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute.

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