Chemotactic selection of frozen-thawed stallion sperm improves sperm quality and heterologous binding to oocytes.

The successful use of assisted reproduction techniques (ART) depends in part on the sperm physiological status. Several sperm selection procedures have been applied to improve quality of sperm population when using the ART. There has previously been development of a Sperm Selection Assay (SSA) for humans which is based on the attraction of capacitated sperm by chemotaxis towards progesterone (P), resulting in an enriched sperm population with an optimal physiological status similar to capacitated spermatozoa, with these cells having very little DNA fragmentation and optimal concentrations of reactive oxygen species (ROS). In the present study, the aim was to adapt the SSA for frozen-thawed stallion semen samples and evaluate the functional status of those sperm selected using the SSA procedure, and to determine whether this enriched sperm population has a greater capacity to bind to the zona pellucida of cattle oocytes. There were experimental conditions developed to conduct the SSA with stallion sperm. Using these conditions, the indexes of induced acrosome reaction, protein tyrosine phosphorylation, mitochondrial membrane potential, mitochondrial and cytoplasmic reactive oxygen species, and number of sperm bound to the zona pellucida of cattle were greater when the sperm population was selected using the SSA. Consistently, the DNA fragmentation and phospholipase C zeta indexes were less for the selected sperm. In conclusion, stallion sperm selected using chemotaxis utilizing the SSA provides a sperm population of greater quality, which when used may improve the outcomes with use of the ART.

Getting to and away from the egg, an interplay between several sperm transport mechanisms and a complex oviduct physiology.

In mammals, the architecture and physiology of the oviduct are very complex, and one long-lasting intriguing question is how spermatozoa are transported from the sperm reservoir in the isthmus to the oocyte surface. In recent decades, several studies have improved knowledge of the factors affecting oviduct fluid movement and sperm transport. They report sperm-guiding mechanisms that move the spermatozoa towards (rheotaxis, thermotaxis, and chemotaxis) or away from the egg surface (chemorepulsion), but only a few provide evidence of their occurrence in vivo. This gives rise to several questions: how and when do the sperm transport mechanisms operate inside such an active oviduct? why are there so many sperm guidance processes? is one dominant over the others, or do they cooperate to optimise the success of fertilisation? Assuming that sperm guidance evolved alongside oviduct physiology, in this review we propose a theoretical model that integrates oviduct complexity in space and time with the sperm-orienting mechanisms. In addition, since all of the sperm-guidance processes recruit spermatozoa in a better physiological condition than those not selected, they could potentially be incorporated into assisted reproductive technology (ART) to improve fertility treatment and/or to develop innovative contraceptive methods. All these issues are discussed in this review.

Improved bovine in vitro embryo production with sexed and unsexed sperm selected by chemotaxis.

Assisted reproductive techniques (ART) have been widely used in farm animals in the last decades. Sexed cryopreserved spermatozoa, ovum pick up, in vitro embryo production and transfer constitute the ART that have revolutionized the dairy industry. However, the efficiency of some of these techniques is still low due in part to sperm quality, which influences fertilization, embryo development and implantation. The Sperm Selection Assay (SSA), based on sperm chemotaxis towards progesterone, provides a sperm subpopulation enriched with spermatozoa that are capacitated, with intact DNA and low level of oxidative stress. Since the SSA selects a sperm subpopulation at optimum physiological state, the application of the SSA may improve the efficiency of the current ART. The aim of this study was to adapt the SSA for unsexed and sexed bovine frozen-thawed semen samples, and then to test whether sperm selection by the SSA improves the cleavage rate of bovine embryos in vitro. The optimal SSA conditions to obtain the higher sperm accumulation percentage given by chemotaxis were the same for both unsexed and sexed semen samples. Thus, sperm accumulation in W2 was significantly higher when: 2 million sperm per mL were placed in W1 (unsexed samples: 12 ±â€¯1%, p = 0.002; sexed samples: 14 ±â€¯3%, p = 0.02); 1 pM progesterone was placed in W2 (unsexed sample: 9 ±â€¯1%, p = 0.009; sexed samples: 11 ±â€¯2%, p = 0.02); and to incubate the SSA device for 10 min (unsexed samples: 17 ±â€¯2%, p = 0.007; sexed samples: 10 ±â€¯1%, p = 0.004). We found that the quality of spermatozoa recovered from W2 in unsexed and sexed semen was enhanced. Thus, the capacitation index was significantly increased (unsexed samples: 1.75 ±â€¯0.1, p = 0.0001; sexed samples: 1.76 ±â€¯0.2, p = 0.004), while DNA fragmentation index was significantly decreased (unsexed samples: 0.33 ±â€¯0.07, p = 0.0003; sexed samples: 0.32 ±â€¯0.04, p = 0.002). Moreover, the cleavage index of oocytes fertilized with either unsexed or sexed SSA-selected sperm was significantly improved (unsexed samples: 3.2 ±â€¯0.4, p = 0.0001; sexed samples: 2.3 ±â€¯0.33, p = 0.03). Thus, we show that the SSA can be used to recruit a bovine sperm subpopulation at optimal functional state regardless of whether the sample is previously sexed, and that this optimal state improves bovine embryo cleavage rate.

Versatile action of picomolar gradients of progesterone on different sperm subpopulations.

High step concentrations of progesterone may stimulate various sperm physiological processes, such as priming and the acrosome reaction. However, approaching the egg, spermatozoa face increasing concentrations of the hormone, as it is secreted by the cumulus cells and then passively diffuses along the cumulus matrix and beyond. In this context, several questions arise: are spermatozoa sensitive to the steroid gradients as they undergo priming and the acrosome reaction? If so, what are the functional gradual concentrations of progesterone? Do spermatozoa in different physiological states respond differentially to steroid gradients? To answer these questions, spermatozoa were confronted with progesterone gradients generated by different hormone concentrations (1 pM to 100 µM). Brief exposure to a 10 pM progesterone gradient stimulated priming for the acrosome reaction in one sperm subpopulation, and simultaneously induced the acrosome reaction in a different sperm subpopulation. This effect was not observed in non-capacitated cells or when progesterone was homogeneously distributed. The results suggest a versatile role of the gradual distribution of very low doses of progesterone, which selectively stimulate the priming and the acrosome reaction in different sperm subpopulations.

Human sperm pattern of movement during chemotactic re-orientation towards a progesterone source.

Human spermatozoa may chemotactically find out the egg by following an increasing gradient of attractant molecules. Although human spermatozoa have been observed to show several of the physiological characteristics of chemotaxis, the chemotactic pattern of movement has not been easy to describe. However, it is apparent that chemotactic cells may be identified while returning to the attractant source. This study characterizes the pattern of movement of human spermatozoa during chemotactic re-orientation towards a progesterone source, which is a physiological attractant candidate. By means of videomicroscopy and image analysis, a chemotactic pattern of movement was identified as the spermatozoon returned towards the source of a chemotactic concentration of progesterone (10 pmol l⁻¹). First, as a continuation of its original path, the spermatozoon swims away from the progesterone source with linear movement and then turns back with a transitional movement that can be characterized by an increased velocity and decreased linearity. This sperm behaviour may help the spermatozoon to re-orient itself towards a progesterone source and may be used to identify the few cells that are undergoing chemotaxis at a given time.

Progesterone sperm chemoattraction may be modulated by its corticosteroid-binding globulin carrier protein.

Progesterone, the main steroidal component secreted by the cumulus cells that surround the egg, chemotactically guides human spermatozoa. The aim of this work was to evaluate whether the carrier protein corticosteroid-binding globulin also participates in the sperm P chemotactic response. By means of videomicroscopy and image analysis, we observed that corticosteroid-binding globulin modulates the chemotactic activity of P, when a solution of corticosteroid-binding globulin + P is at the nanomolar range.

Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex.

Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.

Ca2+ signalling in the control of motility and guidance in mammalian sperm.

Ca2+ signalling in the sperm plays a key role in the regulation of events preceding fertilisation. Control of motility, including hyperactivation and chemotaxis, is particularly dependent upon [Ca2+]i signalling in the principal piece of the flagellum and the midpiece. Here we briefly review the processes that contribute to regulation of [Ca2+]i in mammalian sperm and then examine two areas: (i) the regulation of hyperactivation by [Ca2+]i and the pivotal roles played by CatSpers (sperm-specific, Ca2+-permeable membrane channels) and intracellular Ca2+ stores in this process and (ii) the elevation of [Ca2+]i and consequent modulation of motility caused by progesterone including the ability of progesterone at micromolar concentrations to cause sperm hyperactivation and/or accumulation and the recent discovery that progesterone, at picomolar concentrations, acts as a chemoattractant for mammalian sperm..

Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa.

By means of a videomicroscopy system and a computer image analysis, we performed chemotaxis assays to detect true chemotaxis in human spermatozoa, in parallel to immunohistochemistry detection of progesterone inside the cumulus cells. Progesterone indeed chemotactically guides mammalian spermatozoa at very low hormone concentrations, and the cumulus oophorus could be a potential place for sperm chemotaxis mediated by progesterone in vivo.

Determination of human sperm calcium uptake mediated by progesterone may be useful for evaluating unexplained sterility.

Sperm samples from couples who underwent assisted reproduction were classified according to the World Health Organization (WHO) criteria of concentration, motility, and morphology, in normal and subnormal cases (oligozoospermic, asthenozoospermic, and teratozoospermic). The percentage of spermatozoa that increased [Ca(2+)](i) in response to progesterone (P) was determined by means of flow cytometry. The evaluation of the P-mediated intracellular calcium increase by flow cytometry may be a fast and objective tool for the diagnosis of human sperm samples, especially in cases of unexplained sterility.