Original Article

Toxicol. Res. Vol. 33, No. 4, pp. 315-323 (2017)

https://doi.org/10.5487/TR.2017.33.4.315
Open Access

Environmental Exposure of Sperm Sex-Chromosomes:

A Gender Selection Technique
Ibukun P. Oyeyipo1,2, Michelle van der Linde1 and Stefan S. du Plessis1
1
Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa
2
Department of Physiology, College of Health Sciences, Osun State University, Osogbo, Osun State, Nigeria

Preconceptual sex selection is still a highly debatable process whereby X- and Y-chromosome-bearing
spermatozoa are isolated prior to fertilization of the oocyte. Although various separation techniques are
available, none can guarantee 100% accuracy. The aim of this study was to separate X- and Y-chromo-
some-bearing spermatozoa using methods based on the viability difference between the X- and Y-chromo-
some-bearing spermatozoa. A total of 18 experimental semen samples were used, written consent was
obtained from all donors and results were analysed in a blinded fashion. Spermatozoa were exposed to dif-
ferent pH values (5.5, 6.5, 7.5, 8.5, and 9.5), increased temperatures (37oC, 41oC, and 45oC) and ROS level
(50 µM, 750 µM, and 1,000 µM). The live and dead cell separation was done through a modified swim-up
technique. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent
in situ hybridization (FISH) before and after processing. The results indicated successful enrichment of X-
chromosome-bearing spermatozoa upon incubation in acidic media, increased temperatures, and elevated
H2O2. This study demonstrated the potential role for exploring the physiological differences between X-
and Y-chromosome-bearing spermatozoa in the development of preconceptual gender selection.
Keywords: Sex-chromosomes, Motility, Velocity, Temperature, Hydrogen peroxide, Human

INTRODUCTION tion, although this option is not well-established yet and

therefore, remains quite speculative. However, influencing
The potential capacity to choose the sex of a child has the timing of fertilization with regard to ovulation is one of
intrigued many generations of parents. Sex selection can be the natural methods believed to be effective, while PGD is
accomplished by either applying advanced assisted repro- very costly, highly invasive, and involves associated risks,
duction techniques (ARTs), conducing preimplantation genetic as well as ethical issues (1). Sex selection through timely
diagnosis (PGD), or using cell sorting prior to fertilization. intercourse is based on the respective characteristics of the
In addition, it can also be achieved through natural concep- X- and Y-chromosome-bearing spermatozoa, favoring one
or the other toward reaching and fertilizing the egg (2).
Correspondence to: Stefan S. du Plessis, Division of Medical Physi- The ethicality of sex selection remains an unsettled and
ology, Faculty of Medicine and Health Sciences, Stellenbosch Uni-
versity, Francie van Zijl Drive, Tygerberg 7505, South Africa controversial topic; some countries allow gender selection
Email: [email protected] for medical purposes, while others prohibit any form of sex
selection (3,4). Nonetheless, it is documented that many
List of abbreviations: ANOVA, analysis of variance; ARTs, advanced
assisted reproduction techniques; BSA, bovine serum albumin; families, regardless of their culture or religion, prefer to
PBS, phosphate buffer saline; LIN, linearity; PGD, preimplantation practice gender selection to balance the family gender dis-
genetic diagnosis; SEM, standard error of the mean; STR, straight- tribution (5). Circumventing the ethical issues implies that
ness; SURRG, Stellenbosch University Reproductive Research gender selection must be practiced prior to fertilization.
Group; VAP, average path velocity; VCL, curvilinear velocity; VSL,
Successful separation of X- and Y-chromosome-bearing
straight line velocity; WHO, World Health Organization.
spermatozoa could have great potential, as it could drasti-
This is an Open-Access article distributed under the terms of the cally lower the abortion, infanticide, and abandonment sta-
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted tistics of many countries; hence, there is a need for the
non-commercial use, distribution, and reproduction in any development of ethical, cost effective, and successful meth-
medium, provided the original work is properly cited. ods toward sex selection. To achieve this goal, it is believed

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316 I. P. Oyeyipo et al.

that combining sperm selection with Shettle’s or Whelan’s study was granted by the Health Research Ethics Commit-
methods for the timing of fertilization with regard to ovula- tee 1 (Ethics #: S13/04/068). Semen was collected from
tion would result in highly increased chances of successful healthy donors according to the World Health Organization
preconceptual sex selection (6). (WHO) guidelines (16). In each instance, the semen was
Many differences exist between X- and Y-chromosome- allowed to undergo liquefaction (30 min; 37oC, 95% humid-
bearing spermatozoa, including DNA content, size and den- ity; 5% CO2). The exclusion criteria used in this study were
sity, motility, and surface protein properties (7-9). X-chro- history of smoking and/or illicit drug use, exposure to any
mosome-bearing spermatozoa have been shown to contain occupational or environmental toxicants, and use of medi-
2.9% more DNA than Y-chromosome ones (10). Studies cation. All semen samples included in the study had to con-
have also shown that Y-chromosome-bearing spermatozoa form to normality according to the WHO standards. Samples
swim faster and more progressively than the X-chromo- that did not comply were excluded from the study.
some ones (6). Y-chromosome-bearing spermatozoa have
been shown to have a shorter lifespan, and cannot with- Sperm preparation. A 3% HAMS-BSA solution was
stand exposure to acidic environments or oxidative stress prepared by adding 0.3 g of bovine serum albumin (BSA)
for a very long period of time (8). They are also generally (Sigma-Aldrich, South Africa) to 10 mL of HAMS-F10
considered to be the most fragile among both types of sper- (Sigma-Aldrich). Two hundred microliters of semen were
matozoa. Cui and Matthews concluded that the length, used for sex-chromosome ratio determination, the remain-
perimeter, and area of the spermatozoa’s heads, as well as ing semen was transferred to a conical tube, and 2 mL of
the lengths of the neck region and tail were significantly HAMS-BSA solution was added. The concentrations, motil-
larger and longer in X-chromosome-bearing spermatozoa ity parameters, and viability percentages of the spermato-
than in Y-chromosome ones. Their study demonstrated that zoa were determined after the preparation step, and used to
X-chromosome-bearing spermatozoa are statistically larger indicate sperm parameters at time 0.
than Y-chromosome ones (11).
It has been previously reported that many sperm separa- Fluorescent in situ hybridization (FISH). The ratio of
tion methods have inconsistent results, and for this reason, X- to Y-chromosome-bearing spermatozoa was determined
none of these techniques have gained true acceptance within with 2 color-fluorescent in situ hybridization. This process
the scientific community (12). Therefore, there is still a was performed on the neat sample to establish the natural
need for the development of clinically significant and rec- unchanged sperm sex-chromosome ratio (i.e. before expo-
ognized techniques toward a successful natural strategy for sure), and on the various samples obtained after exposure to
sorting X- and Y-chromosome-bearing spermatozoa. In this the different environments. The FISH protocol was used as
regard, toxicity tests have been proven useful in reproduc- specified by the manufacturer’s instructions (Aczon, Nano
tion (13) and more specifically, spermatozoa, which are Bio Tech, Italy). Briefly, DNA was decondensed and dena-
specialized cells, have been reported to be highly sensitive tured into single strands and slides prepared. The single-
to environmental factors such as temperature and redox stranded DNA was probed with short fluorescence-tagged
state (14,15). Although various studies have reported sev- oligonucleotides that were complementary to regions spe-
eral morphological and functional differences between X- cific to either the X or Y chromosomes. The slides were then
and Y-chromosome-bearing spermatozoa, none of them have incubated in the dye overnight, mounted, and subsequently
proven that these differences are significant enough to allow viewed under a fluorescent microscope. Manual assess-
for the natural separation of spermatozoa into two popula- ment was performed and at least 200 cells were counted.
tions based on these differences. Therefore, the aim of this
study was to separate X- and Y-chromosome-bearing sper- Incubation at varying pH. Phosphate buffer saline (PBS)
matozoa through methods based on their resilience toward (Gibco, Scotland, UK) was used as the incubation medium
exposure to wide ranges of pH, temperature, and H2O2 con- and pH was adjusted to the targeted values of 5.5, 6.5, 7.5,
centration. 8.5, and 9.5 with 1 M NaCl or 1 M HCl. Fresh solutions
were prepared prior to each experiment. After processing,
MATERIALS AND METHODS the pellet was re-suspended in 1.2 mL of HAMS-BSA solu-
tion. A 200-μL volume of the prepared spermatozoa was
Semen sampling. A total of 18 semen samples were then added into each pH solution. After ensuring that the
obtained from healthy volunteers (19~26 years old) taking pH remained stable, sample tubes were incubated for 15 min
part in the Stellenbosch University reproductive research (37oC, 95% humidity; 5% CO2). Two hundred microliters of
group (SURRG) donor program. All the donors were informed sample were used to establish the sperm concentration, motil-
that their spermatozoa would be used exclusively for research ity parameters, and percentage viability at time 0. After
purposes and discarded in an appropriate fashion, after incubation, samples were re-assessed for motility and via-
which they gave their consent. Ethical clearance for this bility parameters. Live cell fractions were isolated by modi-
 Environmental Insults Alter Sex Chromosome Ratio 317

fied swim-up, and the fractions were subsequently stored in collected, they were thawed at room temperature (20 min),
liquid nitrogen (−196oC) until FISH analysis. appropriately pooled, and sex-chromosome determination
was performed via FISH.
Incubation at varying temperature. The prepared • Statistical analysis: Data were presented as mean ±
spermatozoa were divided into 3 aliquots and incubated at standard error of the mean (SEM) and were analyzed using
37oC, 41oC, and 45oC for 25 min. After the incubation period, one-way analysis of variance (ANOVA). Differences dis-
the live cell fractions were isolated using the modified swim- playing p < 0.05 were considered statistically significant. FISH
up protocol, and subsequently stored in liquid nitrogen until data are presented in terms of ratio increase or decrease in
further sex-chromosome determination. absolute percentage difference.

Incubation at varying concentrations of hydrogen RESULTS

peroxide. H2O2 (Sigma-Aldrich, 30% (w/w) in H2O) was
diluted with PBS to yield final concentrations of 2,000 μM, Effect of pH on spermatozoa.
1,500 μM, or 100 μM. Fresh solutions were prepared daily • Sex-chromosome ratio: The effect of pH on the sex-
in order to maintain the integrity of the incubation medium. chromosome ratios of the samples, expressed as the abso-
After processing, the spermatozoa pellet was re-suspended lute changes in the incidence of sex-chromosomes com-
in 1.2 mL HAMS-BSA. A 250-μL volume of the prepared pared to the original values, is shown in Fig. 1. After the
spermatozoa was added to 250 μL of each of the H2O2 solu- 15-min incubation period, there was no change in the X:Y
tions, to give final stimulation concentrations of 1,000 μM, sex-chromosome ratio measured in the neat sample at neu-
750 μM, or 50 μM. A control solution consisting of 250 μL tral pH (7.5) (55% : 45%). When compared to the sex-chro-
spermatozoa mixed with 250 μL PBS was also included. mosome ratio measured in the neat sample, a very acidic
The remaining 200 μL was used to establish the baseline pH (5.5) led to a considerable enrichment in X-chromo-
concentration of the spermatozoa. The solutions were some-bearing spermatozoa, increasing from 55 to 62%,
placed in the incubator (37oC, 95% humidity; 5% CO2) for (12.7% increase). However, the number of Y-chromosome-
25 min, after which live cell fractions were isolated by bearing spermatozoa was enhanced in all the remaining
modified swim-up protocols. These fractions were then samples compared to the neat semen sample, showing per-
stored in liquid nitrogen until the FISH procedure could be centage increases of 8.9% (45% vs. 49%), 4.4% (45% vs.
performed to determine sex-chromosome ratios. 47%), and 8.9% (45% vs. 49%) at pH 6.5, 8.5, and 9.5,
respectively, as shown in Fig. 1 and Table 1.
Semen Analysis. • Sperm motility: Fig. 2A and 2B illustrate the effect
• Motility: The sperm concentration and motility in of incubation at various pH on the percentage of total and
each sample were measured prior to the experiment in order progressively motile spermatozoa in the samples, respec-
to establish normality and baseline values of the sample, tively. The lowest total and progressive motilities were
and thereafter at various time points throughout each exper- observed at pH 5.5, while increases in total and progressive
iment. Sperm concentration and motility were assessed by motilities were observed at pH 9.5.
means of Computer Aided Sperm Analysis (CASA), using • Sperm velocity parameters: The effect of pH on the
the Sperm Class Analyzer (SCA) (Microptics, Barcelona, velocity parameters (VCL, VSL, LIN, and STR) of the sper-
Spain). Several motility parameters were assessed, includ-
ing total motility (%) and progressive motility (%). Veloc-
ity parameters that were measured included curvilinear
velocity (VCL) (μm/s), straight line velocity (VSL) (μm/s),
average path velocity (VAP) (μm/s), linearity (LIN) (%),
and straightness (STR) (%).
• Modified Swim-up protocol: After the incubation
period, the spermatozoa solutions were transferred into a
new conical tube. HAMS-BSA (1 mL) was layered care-
fully on top of the sample, preventing mixing of the solu-
tions. The tube was placed in the incubator at a 45o angle
for 25 min, after which the top 500 μL was carefully
removed and the rest discarded. Motility, viability, and sex-
chromosome ratios were then assessed.
• Sub-zero storage: After swim-up processing, all
samples were frozen in PBS without cryomedium in liquid Fig. 1. Effect of pH on the sex-chromosome ratios in spermato-
nitrogen (−196oC) and stored. Once all the samples had been zoa.

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318 I. P. Oyeyipo et al.

Table 1. X- and Y-chromosome ratios in viable spermatozoa populations after exposure to different environmental conditions
pH incubation
X-chromosome-bearing spermatozoa Y-chromosome-bearing spermatozoa
pH value percentage and percentage change from percentage and percentage change from
‘neat’ sex chromosome in brackets (%) ‘neat’ sex chromosome in brackets (%)
NEAT 55 45
5.5 62 (↑12.7) 38 (↓15.5)
6.5 51 (↑7.2) 49 (↑8.9)
7.5 55 (0) 45 (0)
8.5 53 (↓3.6) 47 (↑4.4)
9.5 51 (↓7.2) 49 (↑8.9)
Temperature incubation
X-chromosome-bearing spermatozoa Y-chromosome-bearing spermatozoa
Temperature percentage and percentage change from percentage and percentage change from
‘neat’ sex chromosome in brackets (%) ‘neat’ sex chromosome in brackets (%)
NEAT 52 48
37oC 52 (0) 48 (0)
41oC 59 (↑13.4) 41 (↓15.5)
45oC 54 (↑3.8) 46 (↓4.2)
Hydrogen peroxide incubation
X-chromosome-bearing spermatozoa Y-chromosome-bearing spermatozoa
H2O2
percentage and percentage change from percentage and percentage change from
concentration
‘neat’ sex chromosome in brackets (%) ‘neat’ sex chromosome in brackets (%)
NEAT 54 46
0,050 μM 54 (0) 46 (0)
0,750 μM 57 (↑5.5) 43 (↓6.5)
1,000 μM 56 (↑3.7) 44 (↓4.3)
↑ and ↓ symbolise increase and decrease, respectively.
Percentage change from ‘neat’ sex-chromosome (Change from the neat value/neat value × 100).

matozoa followed the same trend as the total and progressive 2.264% vs. 37.77 ± 2.264%, p = 0.083).
motilities, indicating that the velocities reach their higher The STR data followed a trend that was very similar to
levels at pH 8.5, while decreasing when the pH becomes that observed for the LIN values. The data indicated that the
either more acidic or more alkaline, as shown in Fig. 2C-2F. greatest STR values were reached at pH 8.5, with statisti-
The VCL reached its maximum at 7.5 and remained rela- cally significant differences observed between pH 8.5 and
tively constant across all pH tested. The VSL data indi- 5.5 (66.89 ± 2.759% vs. 59.25 ± 2.759%, p = 0.001) and
cated a peak at pH 8.5, thereby contrasting with all the other between pH 8.5 and 6.5 (66.89 ± 2.759% vs. 60.54 ± 2.759%,
parameters monitored in this study. There was a statisti- p = 0.006). Decreases in STR were also noted between pH
cally significant difference in VSL between pH 8.5 and 5.5 8.5 and 7.5 (66.89 ± 2.759% vs. 63.53 ± 2.759%, p = 0.132)
(22.58 ± 3.066 μm/s vs. 16.25 ± 3.066 μm/s, p = 0.039) as and between pH 8.5 and 9.5 (66.89 ± 2.759% vs. 62.52 ±
well as between pH 8.5 and 6.5 (22.58 ± 3.066 μm/s vs. 2.759%, p = 0.052).
16.17 ± 3.066 μm/s, p = 0.037).
When considering the linearity of movement of the sper- Effect of temperature on spermatozoa.
matozoa, a peak was observed at pH 8.5. According to • Sex-chromosome ratio: The effects of increased tem-
these results, there were statistically significant differences peratures on the sex-chromosome ratios of the spermatozoa
in the LIN values obtained from spermatozoa placed either are summarized in Table 1 and illustrated in Fig. 3. After
at pH 8.5 or 5.5 (41.02 ± 2.264% vs. 34.90 ± 2.264%, p = 25 min of incubation, at a standard temperature of 37oC,
0.002), between pH 8.5 and 6.5 (41.02 ± 2.264% vs. 35.36 ± there was no change in the sex-chromosome ratio of the
2.264%, p = 0.004), as well as between pH 8.5 and 9.5 sample (X 52 : 52, Y 48 : 48). At 41oC, there was an abso-
(41.02 ± 2.264% vs. 36.47 ± 2.264%, p = 0.017). Further- lute increase of 7% in the incidence of X-chromosome-
more, although not statistically significant, there was a dif- bearing spermatozoa compared to that in the sample prior to
ference in the LIN observed between pH 8.5 and 7.5 (41.02 ± processing (52% vs. 59%), indicating a 13.46% increase,
 Environmental Insults Alter Sex Chromosome Ratio 319

Fig. 2. Effect of pH on motility and velocity parameters of spermatozoa. (A) total motility, (B) progressive motility, (C) curvilinear veloc-
ity (VCL), (D) straight line velocity (VSL), (E) linearity (LIN), and (F) straightness (STR). Significance denoted as: a differs significantly from
b; ab does not differ significantly from a or b.

indicated a statistically significant decrease in the percent-

age of motile cells between 37 and 45oC (74.55 ± 3.883% vs.
55.53 ± 3.883%, p = 0.013). The difference in total motility
between 41 and 45oC (72.28 ± 3.883% vs. 55.53 ± 3.883%, p =
0.022) was also statistically significant, as shown in Fig. 4A.
Progressive motility (Fig. 4B) followed the same trend as
total motility, and declined significantly between 37 and
45oC (35.25 ± 2.719% vs. 11.73 ± 2.719%, p = 0.001) as well
as between 41 and 45oC (39.73 ± 2.719% vs. 11.73 ± 2.719%,
p = 0.0003).
• Sperm velocity parameters: The data obtained for the
velocity parameters after incubating at different tempera-
tures all followed the same trend, as illustrated in Fig. 4C-
Fig. 3. Effect of temperature on sex-chromosome ratios in sper- 4F. Values increased slightly as the temperature increased
matozoa. from 37 to 41oC, before declining significantly as the tem-
perature increased to 45oC.
and at 45oC, the incidence of X-chromosome-bearing sper- The curvilinear velocity increased significantly between
matozoa increased by 2% compared to that in the neat 37 and 41oC (43.43 ± 2.697 μm/s vs. 53.60 ± 2.697 μm/s,
semen sample (52% vs. 54%), representing a 3.84% increase. p = 0.037). There was a statistically significant decrease in
• Sperm motility: Fig. 4A and 4B represent the effect VCL going from 37 to 45oC (43.43 ± 2.697 μm/s vs. 26.10 ±
of temperature on the motility of the spermatozoa. The data 2.697 μm/s, p = 0.004) as well as going from 41 to 45oC

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320 I. P. Oyeyipo et al.

Fig. 4. Effect of temperature on motility and velocity parameters of spermatozoa. (A) total and (B) progressive motility, (C) curvilinear
velocity (VCL), (D) straight line velocity (VSL), (E) linearity (LIN) and (F) straightness (STR). Significance denoted as: a differs significantly
from b and c; b differs significantly from a and c.

μm/s, p = 0.009). A statistically significant decrease in VSL

was also noted between temperatures of 41 and 45oC (21.85 ±
2.180 μm/s vs. 5.85 ± 2.180 μm/s, p = 0.001), as shown in
Fig. 4D.
The results gathered for LIN showed a significant decrease
in linear movement going from 41 to 45oC (40.23 ± 3.111%
vs. 21.93 ± 3.111%, p = 0.004), and between 37 and 45oC
(38.45 ± 3.111% vs. 21.93 ± 3.111%, p = 0.002). There
were virtually no differences in LIN between temperatures
of 37 and 41oC (38.45 ± 3.111% vs. 40.23 ± 3.111%, p =
0.644), as shown in Fig. 4E.
STR results indicated a statistically significant decrease
going from 41 to 45oC (65.10 ± 3.219% vs. 44.58 ± 3.219%,
Fig. 5. Effect of hydrogen peroxide on sex-chromosome ratios
p = 0.0006) and 37 to 45oC (62.73 ± 3.219% vs. 44.58 ±
in spermatozoa. 3.219%, p = 0.001). There was no significant difference in
STR between temperatures of 37 and 41oC (62.73 ± 3.219%
vs. 65.10 ± 3.219%, p = 0.477), as shown in Fig. 4F.
(53.60 ± 2.697 μm/s vs. 26.10 ± 2.697 μm/s, p = 0.0003), as
shown in Fig. 4C. Effect of H2O2 on spermatozoa.
The data obtained for VSL indicated a significant decrease • Sex-chromosome ratio: Upon exposure to H2O2 at
going from 37 to 45oC (16.78 ± 2.180 μm/s vs. 5.85 ± 2.180 50 μΜ, there was no change in the sex-chromosome ratios
 Environmental Insults Alter Sex Chromosome Ratio 321

compared to that in the unprocessed semen samples, as • Sperm velocity parameters: Exposure of spermato-
shown in Fig. 5. At a concentration of 750 μM, an absolute zoa to H2O2 also compromised the velocity parameters of
increase of 3% was observed in the incidence of X-chromo- the cells (Fig. 6C-6F). The same trend could be observed
some-bearing spermatozoa (54% vs. 57%), translating into throughout all the data: velocity decreased steadily and sig-
a percentage increase of 5.5%. Exposure of the spermato- nificantly when going from an exposure to a low H2O2 con-
zoa to 1,000 μM H2O2 still generated enriched X-chromo- centration of 50 μM toward higher concentrations of 750 μM
some-bearing spermatozoa samples, but to a lower extent, and 1,000 μM.
with an absolute increase of 2% compared to the neat The VCL data indicated significant decreases going from
semen (54% vs. 56%), which translates into a percentage a 50 to a 750 μM H2O2 exposure (52.75 ± 4.427 μm/s vs.
increase of 3.7%. 35.23 μm/s, p = 0.031), as well as from 50 to 1,000 μM
• Sperm motility: The effect of H2O2 on motility param- (52.75 ± 4.427 μm/s vs. 25.05 μm/s, p = 0.004), as shown
eters of the spermatozoa are shown in Fig. 6. Both total in Fig. 6C.
(Fig. 6A) and progressive motilities (Fig. 6B) declined sig- VSL was affected similarly, with a significant decrease
nificantly between exposures to 50 and 750 μM (total motil- upon incubation in either 50 or 750 μM H2O2 (18.85 ±
ity, 79.15 ± 9.047% vs. 55.65 ± 9.047%, p = 0.016; progressive 2.353 μm/s vs. 8.30 ± 2.353 μm/s, p = 0.019) and between 50
motility, 53.63 ± 7.697% vs. 22.23 ± 7.697%, p = 0.021) and and 1,000 μM H2O2 (18.85 ± 2.353 μm/s vs. 4.58 ± 2.353
to 50 and 1,000 μM (total motility, 79.15 ± 9.047% vs. μm/s, p = 0.005), as shown in Fig. 6D.
59.63 ± 9.047%, p = 0.033; progressive motility, 53.63 ± The results obtained for LIN and STR indicated a signifi-
7.697% vs. 16.63 ± 7.697%, p = 0.011). There were no sig- cant decrease going from 50 to 1,000 μM H2O2 incubation
nificant differences in any of the motility parameters between (LIN, 35.68 ± 4.017% vs. 16.40 ± 4.017%, p = 0.013; STR,
exposures to either 750 or 1,000 μM. 58.60 ± 4.304 vs. 31.35 ± 4.304, p = 0.004), while STR also

Fig. 6. Effect of hydrogen peroxide on motility and velocity parameters of spermatozoa. (A) total motility, (B) progressive motility, (C)
curvilinear velocity (VCL), (D) straight line velocity (VSL), (E) linearity (LIN), and (F) straightness (STR). Significance denoted as: a differs
significantly from b; ab does not differ significantly from a or b.

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322 I. P. Oyeyipo et al.

decreased significantly going from 50 to 750 μM H2O2 ing that after ovulation, as the pH in the female reproduc-
(58.60 ± 4.304% vs. 38.83 ± 4.304%, p = 0.018), as shown tive tract rises, Y-chromosome-bearing spermatozoa will be
in Figs. 6E and 6F. able to survive better. This increased motility indicates that
Y-chromosome-bearing spermatozoa will reach and fertil-
DISCUSSION ize the oocyte earlier if intercourse occurs a day after ovula-
tion. Higher numbers of X-chromosome-bearing spermatozoa
Current methods employed to perform sperm separation were observed upon exposure to increased temperatures in
are based on the hypothetical existence of fundamental and this study. The control temperature of 37oC had no influ-
physiological differences between X- and Y-chromosome- ence on sex-chromosome ratios, but when the temperature
bearing spermatozoa, as well as on the assumption that was elevated to 41oC, there was a considerable increase in
these differences are significant enough to enable separa- the incidence of X-chromosome-bearing spermatozoa in the
tion. In the present study, the exposure of sperm samples to sample. This increase can be attributed to the Y-chromo-
various pH and temperature ranges, as well as H2O2 concen- some-bearing spermatozoa not being able to withstand this
trations, allowed for enrichment of the viable sperm popula- rise in temperature due to their fragile nature. At 45oC, the
tion with X-chromosome-bearing cells. It was found that at X-chromosome-bearing spermatozoa were still more numer-
an acidic pH (5.5), more X-chromosome-bearing spermato- ous than the Y-Chromosome ones, although the difference
zoa were able to thrive, while more physiological and was not as marked as when incubated at 41oC. Accord-
alkaline pH were more favorable for the survival of Y-chro- ingly, the percentages of total and progressively motile cells
mosome-bearing spermatozoa. This may indicate that the decreased significantly at 45oC. At 41oC, the motility of the
larger size of the X-chromosome-bearing spermatozoa, com- spermatozoa improved, especially the velocity parameters,
pared to that of the Y-chromosome-bearing spermatozoa, probably due to the spermatozoa starting to become hyper-
may allow for an increased cytoplasmic volume (8), which activated at this temperature.
in turn could lead to higher levels of intracellular proteins The effect of H2O2 on the sex-chromosome ratio followed
and phosphates that are able to act as intracellular buffering the same trend as the one observed during the temperature
components, ultimately enabling the X-spermatozoa to sur- incubation experiments, with X-chromosome-bearing sper-
vive in acidic pH environments. However, the increases in matozoa numbers increasing in both the moderate (750 μM)
Y-chromosome-bearing spermatozoa at pH 8.5 and 9.5 indi- and high (1,000 μM) H2O2 fractions. There were no changes
cate the existence of optimal pH ranges for the isolation of in sex-chromosome ratio or viability of the spermatozoa
Y-spermatozoa. Interestingly, all the neat samples had higher after incubation with 50 μM of H2O2. The ability of the X-
ratios of X- to with Y-chromosome-bearing cells. This may chromosome-bearing spermatozoa to survive from expo-
be due to the fact that X-chromosome-bearing cells are more sures to 750 μM and 1,000 μM of H2O2, suggests that these
abundant and viable within the epididymis due to their cells may have more sophisticated intracellular protection
robust nature. mechanisms against hostile environments, since their chro-
The motility data obtained on X- and Y-chromosome-bear- matin content is about 3% denser. It is hypothesized that an
ing spermatozoa enriched samples suggest that the samples increased intracellular storage of antioxidants may take
with higher counts of Y-chromosome-bearing spermatozoa place, or that the membranes of the X-chromosome-bear-
(at pH 8.5 and 9.5) present increased percentages of total ing spermatozoa might be more resistant to the external
and progressively motile spermatozoa. The results gathered environment, which could be resulting from differences in
for the velocity parameters indicate that a pH of 8.5 yields surface charges and/or surface protein properties between
the fastest swimming post-processed spermatozoa in terms the X- and Y-chromosome-bearing spermatozoa. Motility
of VSL, LIN, and STR. This suggests that upon deposition results (total, progressive, and velocity parameters) show a
into the vaginal regions after ejaculation, where pH often significant decrease in the measured parameters when
reaches levels below 4, Y-chromosome-bearing spermato- exposed to 750 μM and 1,000 μM, and the decrease in Y-
zoa might not be able to remain viable for long enough to chromosome-bearing spermatozoa within the samples is
reach the cervical os. The decrease in pH is usually due to possibly due to an overall increase in intracellular ROS
the estrogen surge occurring just prior to ovulation, previ- level. However, incubation with 50 μM H2O2 showed bene-
ously described in the female reproductive tract (17), which ficial effects on the motility parameters of the spermatozoa,
favors the survival of X-chromosome-bearing spermato- especially in terms of velocity-related parameters. The over-
zoa. This is in accordance with Shettle’s method of sex pre- lapping observations made after exposures to either 750 μM
selection, which states that in order to conceive a girl, or 1,000 μM H2O2 concentrations indicated similar deleteri-
intercourse should take place 2-3 days prior to ovulation (6). ous effects produced by these elevated concentrations on
This study also showed that the linear progression was the motility of the spermatozoa. The results of this study
significantly higher at pH 8.5, at which Y-chromosome- indicate that the differences in viability between X- and Y-
bearing spermatozoa were more numerous, thereby suggest- chromosome-bearing spermatozoa after exposure to differ-
 Environmental Insults Alter Sex Chromosome Ratio 323

ent environmental conditions represent a real potential toward 11, 2577-2578.

improving the chances of selecting a preferred gender during 6. Shettles, L.B. and Rorvik, D.M. (2006) How To Choose The
conception. Sex Of Your Baby: Fully Revised And Updated (Rev Upd
This study confirmed once again the existence of differ- edition). Broadway.
7. Gledhill, B.L. (1988) Selection and separation of X- and Y-
ences in viability between X- and Y-chromosome-bearing
chromosome-bearing mammalian sperm. Gamete Res., 20,
spermatozoa, indicating optimal enrichment of X-chromo-
377-395.
some-bearing spermatozoa through incubation in acidic 8. Cui, K.H. (1997) Size differences between human X and Y
media or at increased temperatures, which could represent spermatozoa and prefertilization diagnosis. Mol. Hum. Reprod.,
an interest in the field of ART. The potential to increase the 3, 61-67.
ratio of X-chromosome-bearing spermatozoa even further 9. Gledhill, B.L. and Edwards, R.G. (1993) Can spermatozoa be
by combining these methods requires further investigation. typed? in Preconception And Preimplantation Diagnosis Of
Human Genetic Disease (Edwards, R.G. Ed.). Cambridge Uni-
ACKNOWLEDGMENTS versity Press, Cambridge, UK, pp. 203-231.
10. Johnson, L.A. (1994) Isolation of X- and V-bearing sperm for
The authors are grateful to the Faculty of Medicine and sex preselection in Oxford Reviews of Reproductive Biology,
Vol. 16 (Charlton, H.M. Ed.). Oxford University Press,
Health Sciences, University of Stellenbosch, for the post-
Oxford, UK, pp. 303-326.
doctoral fellowship awarded to the first author, and also to
11. Cui, K.H. and Matthews, C.D. (1993) X larger than Y. Nature,
the Harry Crossley Foundation (South Africa) for funding 366, 117-118.
this research. 12. Flaherty, S.P. and Matthews, C.D. (1996) Application of mod-
ern molecular techniques to evaluate sperm sex selection
CONFLICT OF INTEREST methods. Mol. Hum. Reprod., 2, 937-42.
13. Jeon, H.L., Yi, J.S., Kim, T.S., Oh, Y., Lee, H.J., Lee, M.,
None declared by all authors. Bang, J.S., Ko, K., Ahn, I.Y., Ko, K., Kim, J., Park, H.K., Lee,
J.K. and Sohn, S.J. (2017) Development of a test method for
Received June 5, 2017; Revised July 6, 2017; Accepted July the evaluation of DNA damage in mouse spermatogonial stem
17, 2017 cells. Toxicol. Res., 33, 107-118.
14. Ahmad, G., Agarwal, A., Esteves, S.C., Sharma, R., Almasry,
M., Al-Gonaim, A., AlHayaza, G., Singh, N., Al Kattan, L.,
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