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© Springer Nature Singapore Pte Ltd. 2017
A. Kumar, M. Sharma (eds.), Basics of Human Andrology,
DOI 10.1007/978-981-10-3695-8_11
S. Gupta, PhD (*) • A. Kumar, MD, FAMS
Department of Reproductive Biology, All India Institute of Medical Sciences,
New Delhi, India
e-mail: surabhi72@rediffmail.com
11
The Human Semen
Surabhi Gupta and Anand Kumar
11.1 Introduction
Human semen is a protein-rich body fluid produced by the male reproductive
organs. It is a complex cell suspension in a fluid containing an array of heteroge-
neous substances produced by different male reproductive glands like the testis,
epididymis, seminal vesicles, prostate, Cowper’s gland (bulbourethral) and glands
of Littre (periurethral glands). Its main function is to act as a buffered, nutrient-rich
medium which transports the sperm through the male reproductive tract into the
female reproductive tract.
During coitus, a heterogeneous ejaculate is deposited in the female tract.
This is because the accessory sex glands discharge their secretions by contract-
ing in an organ-specific sequence during emission/ejaculation. This ensures that
the various components of semen are delivered in sequential order. The order of
Learning Objectives
Physical properties
Composition
Fructolysis
Coagulation and liquefaction
164
secretion and the relative contribution of each gland are listed in Table 11.1. The
initial secretion, known as pre-ejaculate, comprises of secretions from the
Cowper’s and Littre glands. This mucinous secretion lubricates the urethra and
neutralizes any traces of residual acidic urine. The next fraction results from the
simultaneous contractions of the epididymis and prostate. It contains the maxi-
mum concentration of sperm along with epididymal and prostatic secretions.
The final and largest fraction of the ejaculate is contributed by the seminal
vesicles.
Two fates exist for the semen components that remain in the male tract after
ejaculation: (i) passive resorption by surrounding tissue or (ii) expulsion during
urination (Prins and Lindgren 2015).
11.2 Physical Properties
The volume of a typical human ejaculate is about 3 ml although it can range
from 2 to 5 ml (Owen and Katz 2005). Normal semen is a greyish, opalescent
fluid with a density between 1.043 and 1.102 g/ml. The colour may appear
whitish due to the presence of high number of sperm or leukocytes. If red
blood cells are present (hemospermia), the colour may appear reddish brown
(WHO 2010).
Semen is slightly alkaline which helps in neutralizing the acidic environment
of vagina. The pH measured can vary from 7.2 to 7.8 depending on the time
elapsed since ejaculation. Decrease in pH of whole semen over time is attributed
to fructolysis and production of lactic acid. However, semen has a buffering
capacity much higher than other body fluids. This buffering capacity is contrib-
uted by bicarbonate/carbon dioxide (HCO3/CO2), high protein content and low
molecular weight compounds like citrate, pyruvate and phosphate (Wolters-
Everhardt et al. 1987). Another peculiar property of semen is its high osmolarity
which is due to the presence of high concentration of organic components rather
than inorganic ions.
Table 11.1 Sequence of secretions during ejaculation
Order of
secretion Contributing gland
% of total ejaculate
volume
Ist Cowper’s gland (bulbourethral) and glands of Littre
(periurethral)
1–5
IInd – A Testis/Epididymis 5–10
IInd – B Prostate 20–30
IIIrd Seminal vesicles 65–75
S. Gupta and A. Kumar
165
11.3 Composition
The components of semen can be divided into ‘cellular’ and ‘acellular’ components
(see Fig. 11.1). The acellular component, obtained after removal of the cells by cen-
trifugation, is termed as seminal fluid and comprises >90% of the semen volume.
11.3.1 Cellular Components
An average human ejaculate has about 100 million sperm/ml though they contribute
less than 1% of the ejaculate volume (Prins and Lindgren 2015). The total number
of sperm per ejaculate correlates with the length of abstinence as well as the testicu-
lar volume (Schwartz et al. 1979; Cooper 2010). Detailed description of sperm is
given in the subsequent chapters, The Sperm and Sperm Function Tests.
The other cellular components of semen are epithelial cells of the urogenital
tract, leukocytes and even spermatogenic cells. The presence of immature germ
Cellular component
Semen
Testis
Epididymis
Seminal vesicles
Prostate
Cowper’s gland
Mature sperm
Epithelial cells of
urogenital tract
Round cells
– Leukocytes
– Glycerophosphocholine (GPC)
– Neutral α-glucosidase
– Free L-carnitine
– Fructose
– Semenogelin
– Prostaglandins
– Zinc (Zn+2)
– Citrate
– Mucin
– Prostate Specific Antigen (PSA)
– Immature germ cells
Acellular component
Secretions from:
Fig. 11.1 Composition of human semen
11 The Human Semen
166
cells in the semen may indicate testicular damage or defective spermatogenesis,
while the presence of leukocytes may be suggestive of inflammation in the acces-
sory glands (WHO 2010).
A simple microscopic examination of semen is not able to differentiate between
leukocytes and spermatogenic cells, which are collectively labelled as ‘round cells’
(Johanisson et al. 2000). Most human ejaculates show the presence of leukocytes
with a predominance of granulocytes. The leukocytes present in the semen are pre-
dominantly peroxidase-positive granulocytes (polymorphonuclear leukocytes). They
can be easily distinguished from the peroxidase-free multinucleated spermatids by
histochemical staining for peroxidase. However, activated granulocytes, which have
lost their granules, and other leukocytes, e.g. lymphocytes, monocytes and macro-
phages, are peroxidase negative. They can be differentiated by immunostaining for
CD45 which is the common leukocyte antigen (WHO 2010). If the number of leuko-
cytes present in the ejaculate is greater than the threshold value (1.0 × 106
peroxidase-positive cells/ml), it is termed as leukospermia. An increase in the total
number of leukocytes present in the ejaculate correlates with the severity of the
inflammatory condition (Wolff 1995).
11.3.2 Components of Seminal Fluid
Seminal fluid comprises of secretions from the seminal vesicles, prostate, testes,
epididymides and Cowper’s and Littre glands with the greatest molecular content
being provided by seminal vesicles (see Fig. 11.1). Some of the constituents are
found in the serum and are probably exudates from the circulation, but many others
are produced exclusively by the reproductive organs and are unique to seminal fluid.
Individual seminal fluid constituents are not essential for fertilization as evidenced
by the fact that epididymal/testicular sperm, obtained by testicular sperm extraction
(TESE), can be used for assisted reproductive technology (ART) to achieve normal
fertilization rates in vitro. However, they may be important under normal conditions
for transport/maturation of sperm and greatly enhance the fertilization capacity of
sperm in vivo.
The precise function of all individual constituents of the seminal fluid has not yet
been determined. They are presumed to be important for the sperm function, during
and/or after ejaculation. Qualitative and/or quantitative assessment of specific
semen components can serve as marker of the proper functioning of each accessory
sex gland, for example, measurement of citric acid, zinc and acid phosphatase to
assess the prostatic gland function; fructose and prostaglandins for seminal vesicle;
free-L-carnitine, glycerophosphocholine (GPC); and neutral α-glucosidase for epi-
didymal function.
Human seminal fluid contains a diverse set of molecules ranging from organic
constituents like proteins, peptides, sugars and lipids to inorganic ions like zinc (see
Table 11.2). Average protein concentration of human seminal fluid is 25–55 g/L
with albumin making up about one third of the total protein present (Owen and Katz
2005; Rodriguez-Martinez et al. 2011). Albumin in semen is mainly of prostatic
S. Gupta and A. Kumar
167
origin, but the majority of other proteins present are contributed by seminal vesicles
(Hirsch et al. 1991). Some of the most important components of the human seminal
plasma are listed in Table 11.2 and discussed in subsequent section of the chapter.
11.3.2.1 Originating from Seminal Vesicles
The most important constituents in the seminal vesicle secretions include fructose,
semenogelin and prostaglandins. Fructose serves as the primary energy source for
sperm in semen. It is produced exclusively by the seminal vesicles, and, hence, its
absence in semen is a sign of potential ejaculatory duct obstruction. Semenogelin is
a 52-kDa protein which is involved in coagulation of semen. The cleavage products
of semenogelin formed following liquefaction have biological functions, such as
inhibition of sperm motility and antibacterial activity. The seminal fluid contains
about 15 different prostaglandins, predominantly prostaglandin E. The prostaglan-
dins induce smooth muscle contractions in the female genital tract, thereby helping
in rapid sperm transport independent of sperm motility. Seminal vesicles are also
the major contributor of phospholipids present in semen. The ratio of cholesterol to
phospholipids in the semen is proposed to help stabilize the sperm against tempera-
ture and environmental shock (White et al. 1976). The other proteins secreted by
seminal vesicles include fibronectin, lactoferrin, protein C inhibitor and prolactin-
inducible protein (Rodriguez-Martinez et al. 2011; Drabovich et al. 2014).
11.3.2.2 Originating from Prostate
The major proteins secreted by the prostate include prostate-specific antigen (PSA),
prostatic acid phosphatase (PAP) and cysteine-rich prostate-specific protein-94
(PSP94). PSA is a zinc-binding serine protease of the Kallikrein family, which
hydrolyzes semenogelin leading to liquefaction of the coagulum. PAP is a 102-kDa
glycoprotein dimer with enzymatic activity. The main substrate for PAP in seminal
fluid is phosphorylcholine phosphate. Prostate also produces spermine which gives
semen its unique odour. Spermine has four positive charges and can bind to acidic
or negatively charged molecules like phosphate ions, phospholipids or nucleic
acids. Enzymatic oxidation of spermine by diamine oxidase, which is present in the
Table 11.2 Important constituents of human seminal fluid
Name of the constituent Concentration (mg/ml) Major source
Phosphorylcholine 10.0 Epididymis
Prostate-specific antigen (PSA) 0.5–5.0 Prostate
Citric acid 3.76 Prostate
Spermine 0.5–3.5 Prostate
Prostatic acid phosphatase (PAP) 0.3–1.0 Prostate
Zinc 0.14 Prostate
Fructose 2.0 Seminal vesicles
Total lipids (cholesterol +
phospholipids)
1.85 Seminal vesicles
Prostaglandins 0.1–0.3 Seminal vesicles
11 The Human Semen
168
seminal fluid, yields aldehyde products which are toxic to both sperm and bacteria.
Hence, prolonged exposure of sperm to seminal fluid reduces their fertilization
capability (Folk et al. 1980; Prins and Lindgren 2015).
Concentration of zinc in the normal human seminal fluid is more than 100 times
compared to concentration in serum. It is involved in regulating liquefaction by
binding to semenogelin. It also has an antibacterial activity. Similar to zinc, the
concentration of citrate in the semen is 500–1000 times higher than that in blood. It
is a potent binder of metal ions, and its concentration (20 mM) compares to the
combined concentration of divalent metals (calcium, 7 mM; magnesium, 4.5 mM;
and zinc, 2.1 mM).
11.4 Fructolysis
Due to the high motility of sperm, their energy requirement is very high. The major
energy source for sperm in the semen is fructose which is produced by the seminal
vesicle. Typical concentration of fructose in human semen is 200 mg/dl. To main-
tain a high adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, the
sperm utilize anaerobic glycolysis of fructose termed as fructolysis. The process of
fructolysis has been described in the chapter, Seminal Vesicles. Each fructose mol-
ecule yields 2 lactate ions and 2 hydrogen ions (H+).
A positive correlation exists between the degree of sperm motility and the rate of
fructolysis in human semen (Peterson and Freund 1976). However, immobilization
by spermicidal agent (lipid peroxidase) leads to irreversible loss of fructolytic abil-
ity of the sperm (Mann et al. 1980).
11.5 Coagulation and Liquefaction
Human semen coagulates spontaneously after ejaculation and subsequently lique-
fies within 15–60 min at room temperature. Although the exact mechanism underly-
ing the process of semen coagulation/liquefaction is not clearly understood, it is
believed to be regulated through a series of enzymes, mainly proteases, inhibitory
factors and metal ions (Emami et al. 2008). Components of the semen are stored in
separate glands and get mixed only upon ejaculation. The prostatic secretion con-
taining Zn+2 and zinc-inhibited PSA are mixed with the seminal vesicle-produced
semenogelin proteins and protein C inhibitor (PCI). Since zinc has a higher affinity
for semenogelins in comparison to PSA, it preferentially binds to semenogelins
after ejaculation. This induces a conformational change of semenogelin leading to
formation of an insoluble, fibrous coagulum. Sperm are immobilized in this coagu-
lum. Chelation of zinc ions diminishes the concentration of free Zn+2, thus activat-
ing PSA. Activated PSA cleaves the semenogelins resulting in liquefaction of the
gel and release of motile sperm (Malm et al. 2007). Zinc and PCI are also released
into solution, and these in turn bind to PSA, preventing further undesirable prote-
olysis. The details of the coagulation and liquefaction are shown schematically in
S. Gupta and A. Kumar
169
Fig. 11.2. The coagulation/liquefaction process allows suitable exposure of sperm
to seminal fluid that stimulates motility, increases fertilizing ability and also permits
orderly entry of sperm into the female genital tract (Hafez 1976). Absent or incom-
plete liquefaction process correlates with reduced fertilizing capability (Prins and
Lindgren 2015).
11.6 Future Directions
It is important to understand the salient physical and chemical properties of normal
human semen in order to formulate a standardized semen simulant. This simulant
fluid would be helpful in research related to intravaginal drug delivery for contra-
ceptive and prophylactic drugs (Owen and Katz 2005).
Another area with untapped potential is the use of seminal fluid as a non-
invasive clinical sample to identify biomarkers for infertility as well as reproduc-
tive tract diseases like prostatitis, cancer, etc. Seminal fluid contains many
molecules which are produced by specific male reproductive organs/glands, and,
hence, any pathological condition of these organs would influence the molecular
composition of semen. Discovery of PSA as a marker of prostatic diseases, both
benign prostatic hyperplasia and prostate cancer, is the best example to illustrate
this point.
Key Questions
In what sequence are the various fractions of semen secreted during
ejaculation?
Name the constituents of seminal fluid and discuss their function.
Describe the key steps in the process of coagulation and liquefaction of
human semen.
Prostate
Epididymis
Seminal vesicles
= Sperm = PSA = Zn+2 = Semenogelin
Ejaculation
Coagulation
15–60 min
Liquefaction
Fig. 11.2 Schematic diagram showing the coagulation and liquefaction process of human semen
11 The Human Semen
170
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S. Gupta and A. Kumar
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Chapter
The accessory reproductiveorgans in the male consist of the continuous series of paired ducts that transport sperm from the testis to the urethra (epididymis and ductus deferens, also known as vas deferens) as well as the accessory sex glands-the prostate, seminal vesicles, bulbourethral glands, and glands of Littre. The main functions of the sex accessory tissues collectively are the transport of sperm and the creation of seminal plasma, the medium in which sperm are delivered in the ejaculate. This product of the sex accessory glands, seminal plasma, is a unique mixture with high concentrations of zinc, fructose, citric acid, prostaglandins, polyamines, proteases, and acid phosphatases. The physiologic role of many of these substances is yet to be understood, but it is widely believed that these constituents play an essential role in male fertility by optimizing conditions for sperm transport and increasing their chance of fertilization. Both the development and homeostatic function of the sex accessory tissues are vitally linked to androgen signaling. Androgens and the androgen receptor, although necessary for normal physiology, are also an integral part of disease processes in the prostate including prostate cancer and benign prostatic hyperplasia. Far more attention has been given to the prostate compared to the other relatively disease-free sex accessory tissues because of its highly prevalent diseases and the enormous impact it has on public health. In addition to androgens, other hormones such as estrogen, prolactin, and growth hormone contribute to both physiologic and pathophysiologic processes in the prostate. Environmental substances that disrupt endocrine signaling have also been implicated as a contributing factor in prostate disease. Additional key areas of research include the prostate stem cell as well as the complex signaling that occurs between stroma and epithelia, and how these aspects contribute to development, normal homeostasis, as well as disease. This review of the accessory sex glands will encompass their function, anatomy, hormonal regulation, and development including emerging evidence for perturbations by endocrine disrupting chemicals, all with an emphasis on the prostate gland where most information is available. We will also summarize diseases of the prostate and provide an overview of future directions and research needs.
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Anaerobic fructolysis was studied in human spermatozoa from 1) normal, 2) oligospermic, and 3) necrospermic semen, as well as 4) in normal spermatozoa irreversibly immobilized with a spermicidal agent (lipid peroxide); the rate of fructolysis was determined by measuring the amount of L-(+)-lactic acid produced during anaerobic incubation in fructose-supplemented sperm suspensions with identical concentrations of spermatozoa and fructose. Motile spermatozoa from normal semen produced lactic acid at a steady rate for at least 2 hours at 37 C, but when immobilized with peroxidized linoleic acid they lost rapidly and irreversibly all fructolytic ability. There was no substantial difference in the rates of anaerobic fructolysis between sperm suspensions prepared 1) from six individual specimens of normal semen and 2) from pooled ejaculates of ten oligospermic patients. In three of a group of four infertile men diagnosed as necrospermic, the immotile spermatozoa failed to produce lactic acid from fructose. In the fourth individual, the spermatozoa, although immotile at the time of testing, were able to convert fructose to lactic acid, but at a reduced rate; this patient's semen has been examined periodically over the last three years and has contained mostly immotile spermatozoa, but a few times motility was definitely observed, especially after treatment with caffeine. The authors conclude from their results that necrospermia may be associated with diverse metabolic defects, one of them being loss of fructolytic ability by human spermatozoa.
Article
Semen is a heterogeneous and complex cell suspension in a protein-rich fluid with different functions, some of them well known, others still obscure. This paper reviews, comparatively, our current knowledge on the growing field of proteomics of the SP and its relevance in relation to the in vivo situation, for the sake of reproductive biology, diagnostics and treatment. Ejaculated spermatozoa, primarily bathing in cauda epididymal fluid, are (in vitro) bulky, exposed to most, if not all, secretions from the accessory sexual glands. In vivo, however, not all spermatozoa are necessarily exposed to all secretions from these glands, because sperm cohorts are delivered in differential order and bathe in seminal plasma (SP) with different concentrations of constituents, including peptides and proteins. Proteins are relevant for sperm function and relate to sperm interactions with the various environments along the female genital tract towards the oocyte vestments. Specific peptides and proteins act as signals for the female immune system to modulate sperm rejection or tolerance, perhaps even influencing the relative intrinsic fertility of the male and/or couple by attaining a status of maternal tolerance towards embryo and placental development. Proteins of the seminal plasma have an ample panorama of action, and some appear responsible for establishing fertility.
Article
The within-subject variability of the semen sperm count (n), volume (v) and the total number of spermatozoa (N) was studied on 220 ejaculates from 36 normal subjects after an abstinence of 7 days or less. For each of the three variables, the within-subject standard deviation, sigma, was practically proportional to the mean, mu; the common value of the coefficient of variation sigma/mu for all subjects was very high: 0.39 for n, 0.28 for v and 0.55 for N. The 95% confidence intervals based on a single ejaculate were asymmetrical and very large, the lower and upper limits being respectively 0.5 x n and 2.3 x n; 0.7 x v and 1.8 x v; 0.4 x N and 2.9 x N. The three semen characteristics for a given subject were highly correlated with length of abstinence: for an increase in abstinence of 1 day there were mean increments of 13 x 10(6)/ml for n, 0.4 ml for v, and 87 x 10(6) for N.
Article
To address the consistent finding of asthenospermia in spinal cord injured men we compared the biochemical constituents of antegrade fractions of electroejaculates of 6 such patients with the manual ejaculates of 6 volunteers. Semen samples in each group were analyzed for 19 biochemical parameters, pH and osmolality. Organic components included triglycerides, glucose, fructose, uric acid, creatinine, urea, total protein, albumin and cholesterol. Metabolic enzymes, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase, lactate dehydrogenase and alkaline phosphatase, were measured. Inorganic constituents included chloride, sodium, potassium, zinc and phosphorous. Although not significant, higher levels of blood urea nitrogen and creatinine were demonstrated in most electroejaculates suggesting urinary contamination of the antegrade specimens. In electroejaculates significantly lower levels (p less than 0.05) of fructose, albumin, GOT and alkaline phosphatase as well as significantly higher levels (p less than 0.05) of chloride were noted. No significant difference in osmolality or pH was found. Moreover, in the electroejaculates the levels of glucose, uric acid and all inorganic constituents approached their corresponding levels in serum. We conclude that biochemical abnormalities of the seminal plasma may contribute to seminal dysfunction of spinal cord injured men and may result from neurological injury to the accessory sex glands or from the electroejaculation procedure itself.
Article
The quantitative contribution of several components to the BC of human semen has been investigated. The role of spermatozoa is negligible (less than 2%). Both the high-molecular components (proteins) and the HCO3-/CO2 system contribute about 25% to the BC. Therefore, about 50% of the BC of semen must be due to low molecular weight components other than HCO3-/CO2.
Article
To analyze the data available on the biologic significance of white blood cells (WBC) in semen of infertility patients. The relevant literature was reviewed. It is not possible to identify reliably WBC by conventional sperm staining techniques. The peroxidase method is sufficient for quantification of granulocytes, but immunocytology is the gold standard for the detection of all WBC populations in semen. Granulocytes are the most prevalent WBC type in semen (50% to 60%), followed by macrophages (20% to 30%) and T-lymphocytes (2% to 5%). The prevalence of leukocytospermia (> 10(6) WBC/mL semen) among male infertility patients is approximately 10% to 20%. There is controversy on the significance of WBC in semen. Whereas some authors did not observe sperm damage in the presence of leukocytospermia, others have found evidence that WBC are significant cofactors of male infertility: [1] seminal WBC numbers were higher in infertility patients than among fertile men; [2] leukocytospermia was associated with decreased sperm numbers and impaired sperm motility; [3] WBC damaged sperm function and hamster ovum penetration in vitro and were important prognostic factors for IVF-ET failure. Because of absence of clinical symptoms, the origin of WBC is difficult to determine. Normally, most WBC appear to originate from the epididymis because vasectomized men show very few WBC in semen. On the other hand, leukocytospermic samples show low citric acid levels, pointing to asymptomatic prostatitis as a source of WBC in semen. Surprisingly, approximately 80% of leukocytospermic samples are microbiologically negative. In some cases Chlamydia trachomatis might have triggered a persistent inflammatory reaction leading to leukocytospermia. Sperm damage by WBC can be mediated by reactive oxygen species, proteases and cytokines. Furthermore, genital tract inflammation facilitates the formation of sperm antibodies. As seminal plasma has strong anti-inflammatory properties and because there is only short contact between sperm and WBC in prostatitis and seminal vesiculitis, inflammations of the epididymis and testis are likely to have the largest impact on sperm. There is ample evidence that WBC can affect sperm function. Further studies are needed to define cofactors that increase or decrease the risk of sperm damage by WBC.