Archives of Andrology

Journal of Reproductive Systems

ISSN: 0148-5016 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/iaan19

Fluorescence Spectra from Human Semen and

Their Relationship with Sperm Parameters

T. Amano, K. Kunimi & M. Ohkawa

To cite this article: T. Amano, K. Kunimi & M. Ohkawa (1996) Fluorescence Spectra from Human
Semen and Their Relationship with Sperm Parameters, Archives of Andrology, 36:1, 9-15, DOI:
10.3109/01485019608987879

To link to this article: https://doi.org/10.3109/01485019608987879

Published online: 09 Jul 2009.

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 FLUORESCENCE SPECTRA FROM HUMAN
SEMEN AND THEIR RELATIONSHIP
WITH SPERM PARAMETERS

T. AMANO
K. KUNIMI
M. OHKAWA
Department of Urology, School of Medicine,
Kanazawa University, Kanazawa, Japan

Fluorescence spectra of human semen, seminal plasma, and spermatozoa were investigated. A total
of 5 1 semen samples were obtained from 45 men, including 40 suspected subfertile men and 5
normal volunteers. Fluorescence spectra excited at 488 nm for each component in 10 mM phosphate-
buffered saline (PBS) were scanned in a range of 500 to 700 nm. Under these conditions, emission
peaks from each component were observed at 622 nm.The intensity of emission peaks from sperma-
tozoa at 622 nm correlated strongly with the concentration of spermatozoa (r = ,837, p = .0001).
Sperm motility also correlated significantly with fluorescence intensity emission peaks from sperma-
tozoa (r = ,369, p = ,019) and semen plasma (r = ,356, p = ,024). To clarify the origin of emission
peak of 622 nm with 488 nm excitation, some components in seminal plasma were dissolved in PBS
and the fluorescence spectra were measured. The standard single solutions in PBS, such as citric
acid, zinc, calcium, and riboflavin (vitamin BJ, did not yield an emission peak of 622 nm with 488
nm excitation. However, blood plasmas and albumin revealed similar fluorescence peaks at 622 nm.
Thus, the fluorescence spectra may originate from some protein complex.

Keywords fluorescence spectra, human semen, seminal plasma, spermatozoa

Seminal analysis is indispensable, and sperm concentration, total sperm count, and motility
are important factors to be considered in male infertility patients. However, it is valuable to
develop a new procedure to illuminate the character of seminal fluid in detail. Recent develop-
ments in photobiology, including fluorescence spectra, have facilitated qualitative and quanti-
tative analysis of a variety of materials. For example, hematoporphyrin derivative levels in
mice tissues were quantified by fluorescence using a fluorescence spectrophotometer [ 11.
In the present study, we examine fluorescence spectra from human semen, seminal plasma,
and spermatozoa as a method to analyze human semen. The objective of this study is to

We thank Norio Miyoshi (Department of Pathology, Fukui Medical School) for his continuous help. This work
was supported in part by a Grant-in-Aid for Scientific Research (C-06671580) from the Ministry of Education,
Science and Culture, Japan.
Address correspondence to Toshiyasu Ammo, Department of Urology, School of Medicine, Kanazawa Univer-
sity, Takara-machi, 13-1, Kanazawa 920, Japan.

ARCHIVES OF ANDROLOGY 36:9-15 (1996) 9

Copyright 8 1996 Taylor & Francis
0148-5016/96 $10.00 + .OO
10 T. Amano et al.

evaluate fluorescence spectra patterns and the intensity of emission peak, in comparison with
conventional seminal analytic parameters. In addition, the components of fluorescence spectra,
which may contribute to improve semen quality, are inspected.

MATERIALS AND METHODS

Subject. A total of 5 1 semen samples were obtained from 45 men, including 40 suspected subfertile
men and 5 normal volunteers. The mean age of the 45 men was 32.6 years (range 2&57 years).

Preparation of Sperm. Semen was kept for 30 min at room temperature. After liquefaction, ejacu-
lates were analyzed for volume, sperm count, sperm per ejaculate, and percentage of sperm motility. The
semen was centrifuged at 3000 rpm for 10 min. The supernatant was prepared as semen plasma sample.
The pellet was resuspended in an equal volume of seminal plasma with 10 mM phosphate-buffered
saline (PBS; Sigma Chemical Company, St. Louis, MO, USA), pH 7.4. The resuspension was washed
twice, and the final resuspension in PBS was used as the spermatozoa sample. Our preliminary data
revealed that the same fluorescence pattern and intensity were obtained from semen stored at -2O"C, and
thus some samples were frozen at -20°C until the measurement.
The single solutions dissolved in PBS, such as citric acid, zinc, calcium, riboflavin (vitamin B2),
blood plasma, and human albumin were also prepared to compare the fluorescence pattern. The concen-
trations of citric acid, zinc, calcium, and albumin were 516, 16, and 37 mgidL and lO.l%, respectively,
which are the average values in Japanese semen [7].

Measurement of Fluorescence Spectra. A 0. I-mL sample of each fraction (semen, seminal plasma,
and spermatozoa) was placed into a 10 x 10 x 48-mm acryl cuvette (Sarstedt, Germany). Then 1.9 mL
of PBS was added and measurements of total 2-mL samples were carried out on a fluorescence spectro-
photometer (Type 850, Hitachi Co., Tokyo, Japan). The fluorescence emission patterns from 500 to 700
nm were recorded as excited by 488 nm from a Xe lamp. The fluorescence intensity with excitation at
488 nm and emission at 622 nm, which was the emission peak from each sample, was compared. The
single solutions in PBS, such as citric acid, zinc, calcium, riboflavin, blood plasma, and human albumin,
were examined by the same method.

Statistical Analysis. Data were analyzed by simple line regression.

RESULTS
The typical patterns of fluorescence from semen fraction samples are shown in Figure 1.
The fluorescence emission peaks were obtained at 622 nm with excitation at 488 nm from
semen, semen plasma, and spermatozoa. But PBS did not yield fluorescence under these con-
ditions. Fluorescence spectra from semen were similar to those from seminal plasma, and peak
intensity at 622 nm of seminal plasma was 0.94 (range 0.42-1.76) times higher than that of
semen. The intensity of the emission peaks from spermatozoa was 0.10 (range 0-0.62) times
lower than that from seminal plasma.
Sperm concentration correlated significantly with fluorescence intensity emission peak at
622 nm from spermatozoa (r = 337, p = .OOOl) (Figure 2). Motility of 40 samples, excluding
11 azoospermia patients, also correlated significantly with fluorescence intensity emission peak
at 622 nm from spermatozoa (r = .369, p = .019) (Figure 3) and semen plasma (r = .356, p :=
.024) (Figure 4). The single solutions dissolved in PBS of citric acid, zinc, and calcium did not
yield an emission peak of 622 nm with 488-nm excitation, as seen in PBS itself. The fluores-
 Fluorescence Spectra from Human Semen I1
7---__-
- -- - eta

-... :Spermatozoa

700
Wavelength (tun)

FIGURE 1 Fluorescence spectra from human semen. The fluorescence emission patterns of
semen, seminal plasma, spermatozoa and PBS from 500 to 700 nm were scanned excited by 488
nm of Xe lamp. The fluorescence intensity with excitation a t 488 nm and emission at 622 nm was
the emission peak from each sample, except PBS.

..................................... .............................................................

0 20 40 60 80 100 120 140 160

Sperm concentration ( x 10 6 Iml)

FIGURE 2 Sperm concentration and fluorescence intensity emission peak a t 622 nm from sper-
matozoa. Sperm concentration correlated significantly with fluorescence intensity emission peak a t
622 nm from spermatozoa (r = 337,p = .0001).
12 T. Amano et al.

604 - f . ' - ' - ' - ' . ' . ' - ' - ' . a

0
50- ~0.369
ii p=o.o19 0
40-
0 0
0
30. i 0

I
-101 - i - , -.
. , . . . ~. - . - . ~.
. . f

-10 0 10 20 30 40 50 60 70 80 90 100

Motility (%)
FIGURE 3 Motility and fluorescence intensity emission peak a t 622 nm from spermatozoa.
Motility, excluding azoospermia patients, correlated significantly with fluorescence intensity emis-
sion peak a t 622 nm from spermatozoa ( r = .369, p = .019).

2 5 0 ~- ; - ' - ' ~ a '

225. t ~0.356 0
200. 1 p-0.024 0

i
50- 0
25: 6
o i c

Motility (98)
FIGURE 4 Motility and fluorescence intensity emission peak a t 622 nm from semen plasma.
Motility correlated significantly with fluorescence intensity emission peak a t 622 nm from semen
plasma (r = .356, p = .024).
 Fluorescence Spectra from Human Semen 13

- :Semen
----- :Blood plasma

.-
-
9
.
:Albumin
:Riboflavin
:PBS
- 2

,---.. E
~

600
Wavelength (nm)

FIGURE 5 Fluorescence spectra from human semen, riboflavin, blood plasma, and albumin.
Riboflavin had a fluorescence peak at 520-530 nm. Blood plasma and albumin had 2 fluorescence
peaks at 520 and 622 nm. The peak from blood plasmas and albumin at 622 nm revealed a
fluorescence pattern similar to that of semen, seminal plasma, and spermatozoa.

cence peak from PBS solution of riboflavin was obtained from 520 to 530 nm; however, no
fluorescence peak around 622 nm was observed. On the other hand, blood plasma and albumin
had two fluorescence peaks at 520 and 622 nm. The second peak from blood plasmas revealed
a fluorescence pattern similar to that from semen, seminal plasma, and spermatozoa at 622 nm
(Figure 5).

DISCUSSION
Luminescence phenomena occur in living reactions, including the reproductive process. For
example, chemiluminescence has been observed during the fertilization of sea urchin eggs [4].
Fluorescence from nonliving organic substances has also been reported [6]. In the present
study, semen, seminal plasma, and spermatozoa fraction in PBS solution were excited at 488
nm by Xe lamp, and emission from 500-700 nm were scanned.
The 488-nm wavelength was selected because peak fluorescence emission intensity at 622
nm was obtained from the preliminary results. Tang et al. [5] reported the fluorescence spectra
from human semen excited by an argon-ion laser operating at 488 nm. But the patterns of the
fluorescence are different from our results. The spectra peaks for different components are
located at 622 nm in our data, and 527-535 nm in the previous report. The difference between
these experiments is the source of excitation (laser radiation and Xe lamp). In spite of these
differences, a strong linear correlation between the intensity of the spectra and the sperm count
14 T. Amano et al.

was observed in both results. Thus, a component (or some components) in spermatozoa can
contribute to the spectra peak at 622 nm.
Sperm motility is another important indicator of semen quality [2]. Although a majority of'
cases of subfertility will remain unexplained, both sperm and seminal fluid factors influence
sperm motility. Sperm factors, such as immotile-cilia syndrome, have been proved to be causes
of asthenozoospermia. The fluorescence spectrum at 5 10-530 nm from spermatozoa was ob-
served mainly from sperm tails in a previous study [5]. Sperm tails, especially the middle
portion, contain the enzymes that contribute to sperm movement [ 5 ] . These enzymes, such as
riboflavin, might be one of the candidates for semen fluorescence at 510-530 nm [5]. Our
results showed that motility correlated significantly with the fluorescence intensity emission
peak at 622 nm from spermatozoa. These results suggested that the intensity of the spectra
from spermatozoa could reflect a part of the sperm tail function, although the peak intensity
wavelength differed in each experiment.
Seminal plasma contains a variety of substances that may be important for the maintenance
of sperm motility [3]. Sperm motility also has a significant linear correlation with the intensity
of the spectra from seminal plasmas. These results suggest that there are some factors in
seminal plasma that contribute to sperm motility. Therefore, sperm motility is influenced both
by factors of spermatozoa and seminal plasmas, and the components of 622-nm emission with
488-nm excitation can activate sperm motility. In other words, strong fluorescence intensity of'
622 nm with 488-nm excitation from spermatozoa and seminal plasma reflects sufficient sperm
motility.
The component responsible for the spectra peak has not been established. It is important to
determine the main components in semen that yield fluorescence spectra. The fluorescence
spectra peaks from human semen components excited by an argon-ion laser operating at 488
nm were 520-530 nm, and riboflavins were considered to fluoresce in a previous report [ 5 ] .
But the fluorescence spectra peaks from human semen components excited by Xe lamp oper-
ating at 488 nm were 622 nm in the present study.
Blood plasma and albumin have spectra peaks at 622 nm, though these spectra patterns are
not identical with those of semen or seminal plasma. It would appear that some substances,
such as some kinds of protein, play an important role in the spectra peak at 622 nm. Further-
more, the main components of fluorescence spectra are stable after being stored at -20°C.
Thus, the components may be rather stable and in part similar to those of blood plasma and
albumin. In hture studies, we are planning to identify the main components in semen elements
that fluoresce at 622 nm. There is a possibility that sperm concentration and motility might be
improved by the administration of these components.

REFERENCES
1. Amano T, Prout GR, Lin CW (1988): Intratumor injection as a more effective means of porphyrin administra-
tion for photodynamic therapy. J Urol 139:392-395.
2. MacLeod J, Gold R (1953): The male factor in fertility and infertility. Fertile Steril 4:lO-33.
3. McConnell JD (1991): Abnormalities in sperm motility: techniques of evaluation and treatment. In: Infertility in
the Man, 2d ed., Lipshultz LI, Howards SS, eds. St. Louis: Mosby-Year Book, pp 254-276.
4. Takahashi A, Totsune-Nakano H, Nakano M, Mashiko S, Suzuki N, Ohma C, Inaba H (1989): Generation of 0;
and tyrosine cation-mediated chemiluminescence during the fertilization of sea urchin eggs. FEBS Lett 246: 117-
119.
 Fluorescence Spectra from Human Semen 15

5 . Tang GC, Oka N, Nagamatsu GR, Alfano RR (1993): Laser fluorescence spectroscopy from human spermato-
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6. Timms RE, Roupas P, Rogers WP (1982): Determination of oxidative deterioration of milk powder and recon-
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