Sperm Collection of Differential Quality Using Density Gradient Centrifugation

Podsumowanie

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Streszczenie

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

Wprowadzenie

In vertebrates, male gametes undergo intense selective pressure; therefore reproductive fitness of a male is pivotal for achieving successful fertilization. Males of any given vertebrate species must be able to produce sperm cells in large quantities and of sufficient quality in order to meet the needs of fertilization. Sperm cells, having both a sperm head and a flagellum, are the most polarized cells in the body. They are also very heterogeneous in quality of sperm (live and dead, morphologically normal and abnormal, and immobile, low mobile and high mobile), which is revealed through the wide variation in reproductive efficiency of the males. The larger the proportion of high-quality sperm, the fewer the number of matings required to successfully fertilize the ovum. However, to achieve fertility, morphologically normal sperm cells rely on propulsive forces generated by their flagella to reach the site of fertilization as well as to penetrate the zona pellucida¹ (ZP; in the case of mammals) or inner perivitelline layer² (IPVL; in the case of birds and reptiles) of the ovum following natural mating or artificial insemination (AI). Determining sperm quality is necessary for use in assisted reproductive technologies³ (ART) and selection of breeding males to be used in AI programs⁴. On the other hand, the success of ART solely relies upon the accurate evaluation of sperm quality. A number of laboratory tests have been developed to determine the functional characteristics of sperm. The most important parameters are sperm morphology, viability, mobility, capacitation (avian sperm do not require capacitation⁵), acrosome reaction (AR; exocytosis and release of a proteolytic enzyme from the acrosome of the sperm head), sperm penetration of ZP or IPVL, and fertilization^{6,7,8,9,10,11}. Measures of fertility alone do not provide an accurate evaluation of the fertilizing ability of a sperm population¹¹. Measures of the several events leading up to fertilization of an egg allow for an appropriate representation of the performance of individual spermatocytes⁷.

The methodology developed for measuring sperm function is primarily species-specific. For example, in avian sperm, viability, mobility and penetration of IPVL are the most common parameters used to assess sperm quality^8,11,12. The number of live sperm in the ejaculate plays a crucial role for the survival of sperm because the presence of a large number of dead sperm in the semen affects the quality of sperm. This enhances the production of reactive oxygen species in the semen and causes oxidative damage to the live sperm¹³. Sperm mobility, the capacity for flagellar movement of avian sperm against resistance at body temperature, is known to play a direct role in bringing about fertilization⁸. It is well established that mobility is positively correlated with fertility and is, therefore, a primary determinant of fertility⁸. However, a mobile sperm must also have the ability to undergo an AR and to penetrate the IPVL¹¹. IPVL penetration assays take account for every sperm that participates in the process of fertilization¹¹.

In the application of ART, ejaculate is usually processed in order to maximize the concentration of high-quality sperm and minimize concentration of low-quality sperm. After collection of semen, the proportion of high-quality sperm can be enriched through sperm separation procedures commonly used in both industry and research practices. Many of these procedures have been developed, all with respective benefits and limitations, but all utilize the heterogeneous nature of sperm to collect only the sperm with high fertilizing ability. These procedures include sperm migration methods, adherence column filtration and density gradient centrifugation (DGC)^{14,15,16,17,18,19,20}. Among the available techniques, DGC has been found to be very simple, repeatable, cost-effective and efficient in isolating the maximum amount of high-quality sperm for use in ART with the goal of maximizing chance of fertilization^14,15. In addition, DGC is not injurious to the sperm cell membrane. In contrast, sperm migration methods collect only progressively mobile sperm^18,19, but the quantity of sperm collected is very low, making it inefficient in collecting large volumes of sperm^18,20. Adherence column filtration is very efficient in filtering highly mobile sperm from semen¹⁷; however, it tends to be injurious to sperm membranes^20,21.

In the DGC technique, the most commonly used substrate for generating the density gradient is Percoll, which consists of colloidal silica particles coated in polyvinylpryrolidone. Percoll density gradient centrifugation (PDGC) can either be continuous or discontinuous but a discontinuous gradient is most commonly used for high yield isolation of highly mobile sperm^13,16,20. In a discontinuous gradient, lower density media floats above higher density media, creating a gradient that increases in density from the top to the bottom of a conical tube. This creates boundaries at the interface between the two media of differing density. The efficiency of PDGC is derived from two factors: 1) the propulsive ability of individual sperm cells and 2) the tendency of sperm cells with high structural integrity to have an increased density. Sperm with higher mobility are better able to cross from lower density media and penetrate into a higher density media. Lower mobility sperm are more likely to become trapped at the boundary created by the interface between media of differing density. Sperm cells with high structural integrity and mobility tend to have a higher density than dead, abnormal or low mobile sperm cells. When centrifugal force is applied in PDGC, this facilitates movement of sperm with different densities to their respective place in the gradient.

In general practice, after PDGC is performed, the soft pellet of sperm with high fertility potential at the bottom of the conical tube is collected, and the remainder is discarded. However, an underutilized advantage of this technique is its ability to separate sperm cells into several groups based on the quality differences. For research purposes, separation of sperm by degree of quality utilizing the PDGC technique allows for study of sperm quality as it pertains to physiologic, metabolomic and proteomic differences. Here, we aim to detail how this technique may be used to separate sperm by quality, as well as demonstrate these differences in quality, using the previously established eosin-nigrosin vital staining for viability, Accudenz assay for mobility, and sperm-IPVL interaction assay for penetrability.

Protokół

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia.

1. Washing using Traditional Centrifugation

1. Prepare phosphate buffer solution (PBS). Add 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄ and 0.24 g of KH₂PO₄ to 800 mL of distilled water (dH₂O). Adjust the pH to 7.4 using 0.1 N HCl and bring the solution to 1 L using dH₂O.
2. Prepare motility buffer. Add 6.5 g of NaCl, 4.5 g of glucose, 0.444 g of CaCl₂ and 11.5 g of N-tris-[hydroxymethyl] methyl-2-amino-ethanesulfonic acid (TES) to 800 mL of dH₂O. Adjust the pH to 7.4 using 1 M NaOH and bring the solution to 1 L using dH₂O.
3. Pipette 0.5 mL of semen into a polypropylene microcentrifuge tube. Add 1.0 mL of PBS and mix gently.
4. Centrifuge at 1,500 x g for 10 min at room temperature (RT) and discard the supernatant. Resuspend the sperm pellet with PBS up to 1.5 mL.
5. Centrifuge at 1,500 x g for 10 min at RT. Resuspend the sperm pellet with motility buffer up to 0.5 mL.

2. Performing the PDGC technique

NOTE: Perform the entire process of PDGC at room temperature.

1. Make 3.0 mL of 1.08 g/mL and 1.07 g/mL Percoll solutions in two separate tubes.
   1. In a clean test tube, add 1.712 mL of the 1.13 g/mL original Percoll to 0.3 mL of 1.5 M NaCl solution. Add 0.988 mL of dH₂O and mix by gentle inversion to make 3.0 mL of a 1.08 g/mL density solution.
   2. In a clean test tube, add 1.482 mL of the 1.13 g/mL original Percoll to 0.3 mL of 1.5 M NaCl solution. Add 1.218 mL of dH₂O and mix by gentle inversion to make 3.0 mL of a 1.07 g/mL density solution.
2. In a clean test tube, dilute 1.0 mL of semen sample 1:2 with 2.0 mL of PBS. Mix gently by pipetting.
3. Pipet 3.0 mL of the 1.07 g/mL density solution into a sterile 15 mL conical tube. Carefully pipet 3.0 mL of the 1.08 g/mL density solution beneath the 1.07 g/mL density solution. Ensure that the two layers do not mix. A long-form (9 in) Pasteur pipette can make this step easier.
4. Pipet 3.0 mL of diluted semen sample overtop the PDG. To ensure that the semen sample does not mix with the PDG, gently tilt the conical tube containing the PDG at a 45° angle. Pipet the sample along the wall of the tube and allow it to flow down the tube and over the PDG.
5. Prepare a blank tube to match the mass of the PDG with overlaid sample. Centrifuge both tubes at 1500 x g for 20 min. Be careful to maintain the discontinuous gradient while transferring the tubes from the bench to the balance and then to the centrifuge.
   NOTE: Do not use the brake at the end of centrifugation.
6. Observe the results. Ensure that three distinct semen layers have formed in the tube, as seen in Figure 1.
7. Aspirate isolated semen layers with a pipette. Collect the top layer of semen first, the middle layer second, and last the hard pellet at the bottom of the tube. Transfer each to a clean and sterile polypropylenemicrocentrifuge tube.
8. Dilute each sample to 1.5 mL with PBS. Centrifuge at 1500 x g for 10 min.
9. Pour off the supernatant. Reconstitute sperm pellet with motility buffer by gentle pipetting.
   NOTE: Alternative densities may be used to suit investigator needs. Determine amounts of ingredients used with the following equation, where v₀ is volume of stock density solution used, v is final volume of solution desired, p is density of final density solution desired and p₀ is the density of the stock density solution:
   
   Always use 0.3 mL of 1.5 M NaCl in preparation of density solutions to match the NaCl concentration of physiological saline.

3. Determining Sperm Quality

1. Calculate the sperm concentration as previously described²².
2. Perform eosin-nigrosin vital staining as previously described¹² with the following modifications:
   1. Prepare 100 µL of sperm solution at a concentration of 1 x 10⁸ cells/mL.
   2. Pipet 50 µL of sperm solution in a polypropylene microcentrifuge tube containing an equal volume of eosin-nigrosin stain. Incubate the mixture for 5 min at room temperature.
   3. Place a 20 µL drop of stained sperm sample at one end of a glass slide and smear uniformly in a manner similar to that used for blood smears. Air-dry the smeared slides at room temperature for 3-5 min.
   4. Observe the smear under microscope. Count the number of live sperm (no stain) and dead sperm (stained pink) and calculate the percentage of live sperm.
3. Perform the Accudenz assay, as previously validated for the chicken sperm, to objectively assess the sperm mobility⁸ with the following modifications:
   1. Pipet 1.0 mL of 6% assay solution into polystyrene cuvettes, as illustrated in Figure 2. Incubate to 41 °C.
      NOTE: 41 °C is used to match the internal temperature of a hen. The incubation temperature should match that of the female reproductive tract of the species being investigated.
   2. Overlay the preheated assay solution with 100 µL of semen sample at a concentration of 5 x 10⁸ cells/mL.
   3. Place the cuvette containing overlaid sperm sample in the spectrophotometer. Record the absorbance value at 550 nm.
4. Perform IPVL-penetration assay as previously described¹¹ with the following modifications:
   1. Cut a piece (0.5 cm x 0.5 cm) of non-germinal disc region of intact IPVL.
   2. Adjust sperm concentration to 4 x 10⁶ cells/mL
   3. Incubate sperm in motility buffer with IPVL in a small glass vial for 15 min at 37 °C, as illustrated in Figure 3.
   4. Immerse the IPVL piece in 3% NaCl to stop the interaction between the IPVL and sperm.
   5. Mount the IPVL piece on a microscope slide and stain with Schiff's reagent for 10 min following fixation with 10% formalin for 20 s.
   6. Observe the IPVL under a microscope for successful sperm penetration holes and count the number of all visible holes per 0.25 mm² at 40X magnification.

Wyniki

The PDGC technique resulted in distinct separation of three layers of sperm by degree of quality across all parameters. Sperm separates into a high-quality layer below the higher density solution, a medium-quality layer between the higher and lower density solution and a low-quality layer above the lower density solution. These differences in quality are evidenced by clear differences in viability (Figure 4), mobility (Figure 5) ...

Dyskusje

Fertility not only determines the profitability of animal production but also acts as a means of natural selection of species for existence. The ultimate function of a sperm cell is to fertilize an ovum. The oviduct of a female selects only those fittest sperm in order to ensure fertilization of the ovum^23,24. In vitro studies have also revealed a close correlation between qualitative sperm traits and fertilization success^4,<...

Materiały

Name	Company	Catalog Number	Comments
Accudenz	Accurate Chemical and Scientific Corporation, Westbury, NY, USA	AN7050
Percoll	Sigma-Aldrich, Corp., St. Louis, MO, USA	P7828
Schiff’s reagent	Sigma-Aldrich, Corp., St. Louis, MO, USA	3952016
TES	Sigma-Aldrich, Corp., St. Louis, MO, USA	T1375
Eosin Y	Sigma-Aldrich, Corp., St. Louis, MO, USA	E4009
Nigrosin	Sigma-Aldrich, Corp., St. Louis, MO, USA	198285
ST 40R Centrifuge	Thermo Scientific, Waltham, MA, USA	75004524
DU 530 Life Sciences UV/Vis Spectrophotometer	Beckman Coulter, Brea, CA, USA		No catalogue is found
Olympus IX 71 Inverted Fluorescence and Phase Contrast Microscope	Olympus America Inc., PA, USA		No catalogue is found