What Are Gametes Good For? Protection against Micro-Organisms
One prerequisite for multi-cellular organisms to survive is to be able to repulse attacks by micro-organisms, their DNA, RNA and proteins and prions. Every individual multi-cellular organism has developed an immunological defence system that is directed towards everything but itself. However, to discriminate foreign cells and micro-organisms from cells belonging to itself it is essential to be unique. The problem to become unique was solved some 600 million years ago with the evolution of a new type of cell division, meiosis, which enabled the formation of genetically unique gametes. The fusion (fertilization) of two genetically unique gametes (the spermatozoon and the oocyte) results in a new individual with a unique genetic constitution. Due to meiotic recombination, every gamete is supplied with one out of four unique DNA molecules for every chromosome pair. In human beings there are 23 pairs of chromosomes, so the number of possible DNA combinations in any gamete is 423, i.e. any gamete achieves 1 out of at least 70 × 1012 combinations (1 out of 70 million millions) of genetic material. At fertilization, gametes from two different individuals fuse and form one new individual; the genetic composition of the child is thus one combination out of 4900 × 1024 possible combinations. A male human produces some 100 million genetically unique lots per day. A woman produces normally one mature oocyte a month, and again each oocyte becomes as genetically unique as any spermatozoon upon when the second meiotic division is completed fertilization.
Thus, the evolution of meiosis and unique gametes was a prerequisite for an individual immune defence, which in turn was a prerequisite for the evolution and survival of multi-cellular organisms exposed to endless attacks of micro-organisms [Reference Kvist1,Reference Graves2].
Outside the body there is no immune system or reproductive tract to protect the gametes. The laboratory must therefore fulfil these functions to protect gametes and embryos. Assisted reproduction in vitro would be impossible if micro-organisms were not actively combatted. Laminar-air-flow benches, sterile and controlled handling and culture media, rooms with controlled air purity, and special clothing for the involved staff are some of the precautions expected for best practice [Reference Mortimer, Cohen and Mortimer3,4]. Sometimes chemical weapons like antibiotics are necessary. Nonetheless, micro-organisms with foreign DNA and RNA can invade our culture media and become incorporated into embryos and thereby future generations [Reference Kvist1].
Every Man Is a Unique Experiment by Nature
In most multi-cellular organisms two types of gonads, ovaries and testes, have developed that produce two different types of gametes: immotile oocytes and motile spermatozoa, both evolved to fuse with each other. Usually (but not always) only one type of gonad exists within an individual. In evolutionary terms the development of two different types of gametes is an important mechanism for achieving the mixture of genes from two different individuals and counteracting the simple fusion of gametes from a single individual. Hence, among animals, the development of the male and female sexes respectively can be seen as nature’s way to improve probabilities for individuals to find an individual with compatible gametes. In plants, access to spermatozoa (pollen) is facilitated via wind, water or other vectors, while in mammals direct contact between the two gamete-bearing individuals is required.
In mammals, some 300 million years ago, the genes controlling sperm production were transferred from one of the two ancient X-chromosomes onto a “shortened X”, today called the Y-chromosome. As a consequence, mammals are heterogametic rather than homogametic, i.e. they have two different types of gametes, one carrying the X chromosome the other the Y.
The Y-bearing organism developed into a mobile, sperm-producing individual. In order for the ‘species’ to survive, the sperm producer must succeed in finding signs of ovulation and be able to deliver spermatozoa for fertilization of the oocytes. In some species of Asian mole, the Y chromosome has disappeared and critical male-determining genes have moved onto a somatic chromosome.
In mammals, the default development (phenotype) is the female development. The development of a fertile man able to react to signs of ovulation requires the selection of a handful of specific male development routes, during embryonic, fetal and childhood development [Reference Neill5,Reference Nieschlag, Behre and Nieschlag6]. Many of these traits are known to be dependent on testosterone and result in the development of (a) the testes, (b) internal male genital structures, (c) external male genitalia, and (d) male sexual identity.
Spermatogenesis is the process by which spermatozoa are produced from spermatogonia in the testis. The light microscopic examination performed during semen analysis aims to give information about the success of spermatogenesis, including the number of spermatozoa, and the success of spermiogenesis by their morphology and motility. A more thorough evaluation of the ejaculate can reveal a variety of disturbances originating in the different steps of spermatogenesis and might shed light on disturbed testicular function or even reveal CIS-cells indicating the presence of early testis cancer [Reference Nieschlag, Behre and Nieschlag6,Reference Holstein, Schulze and Davidoff7].
Already during the embryonic and fetal stage, preparations for spermatogenesis are being made. Immature germ cells from the epiblast migrate from the yolk sac and invade the seminiferous cords (which will become tubules at puberty) in both testes, and start to proliferate up to week 18 in the fetus. The other cells inside the seminiferous cords, the Sertoli cells, also multiply. The somatic Sertoli cell and its spermatogonia could be regarded as a unit for future sperm production. If the migration of germ cells is disturbed, or if the germ cells degenerate, few or no spermatogonia will be left for sperm production and the only cells left would be the Sertoli cells (known clinically as the ‘Sertoli cell only’ syndrome).
1) Renewal of stem cells. There are two types of spermatogonia type A: dark and pale (Figure 2.1). Both belong to the stem cell population and are continuously renewed by mitotic divisions. It is estimated that spermatogonia undergo some 20 mitotic cell divisions a year, so at 35 years of age the spermatogonia have undergone some 400 mitotic cell divisions, whereas the female oocyte rests from embryonic week 10 until ovulation. Thus, hazards linked to cell division events (mutations in the DNA, aneuploidy, mutations and deletions in mitochondrial DNA) are more likely to affect spermatogenesis than oogenesis.
2) Spermatocytogenesis. An important process is an exponential increase in number of spermatozoa; in the human testis two consecutive mitotic divisions prepare for the meiotic cell division. One spermatogonium A pale is recruited for sperm production and undergoes two mitotic cell divisions, resulting in a clone of four spermatogonia B. Each of these then differentiates into four primary spermatocytes. The latter two mitotic cell divisions increase the possible number of spermatozoa by a factor of four. If one mitotic division does not occur, then only half the number of spermatozoa can be produced. It means that missing mitotic divisions could be one cause of low sperm numbers. In rodents, there may be 11 mitotic divisions before the meiotic divisions begin, and in the rhesus monkey there may be five mitotic divisions. Thus, in those species, one A- spermatogonium will theoretically result in 8192 and 128 spermatozoa, respectively, whereas the number of resulting spermatozoa in man is 16 [Reference Ehmcke and Schlatt8].
3) Meiosis. The purpose of this process is to ensure that every spermatozoon achieves (a) a unique combination of DNA and (b) a haploid genome in which the original 23 pairs of chromosomes are reduced to 23 single copies of the DNA. Each of the four primary spermatocytes in a clone undergoes the two meiotic divisions. From eight secondary spermatocytes finally 16 round spermatids are formed.
4) Spermiogenesis. This is the process where the round spermatid transforms (differentiates) into a functional messenger cell called the testicular spermatid that is still attached to the Sertoli cell (Figure 2.2).
5) Spermiation. Initiated by the Sertoli cell, testicular spermatozoa are released from the Sertoli cells, which take up the surplus cytoplasm and membrane from the sperm midpiece (the residual body).
Spermatogenesis Takes Place in the Seminiferous Tubules
A normal tubule has a diameter of about 180 µm, and the diameter is decreased when spermatogenesis is impaired (Figure 2.3). The tubule walls are composed of five layers of myofibroblasts in connective tissue, which cause peristaltic waves of contraction to transport the immotile testicular spermatozoa to the rete testis for further transportation, via the efferent ducts, to the caput of the epididymis. The thickness of the peritubular tissue is 8 µm, corresponding to the size of the neighbouring spermatogonia. A thickened wall is associated with impaired spermatogenesis [Reference Holstein, Schulze and Davidoff7].
The Sertoli cell is the dominating cell in the seminiferous cords and tubules. It is a supporting cell that provides nutrition and mediates paracrine signals for spermatogenesis as well as protection of the developing germs cells from the immune system. The Sertoli cell communicates, via factors and testosterone, with the Leydig cells in the interstitium between the seminiferous tubules. It produces inhibin B, which exerts a negative feedback on the FSH secretion from the pituitary gland. In about half of men with azoospermia, the Sertoli cells produce low levels of inhibin B, resulting in elevated FSH levels in the blood. However, low levels of inhibin B and high levels of FSH do not exclude that focal spermatogenesis can be found at testicular biopsy, making sperm retrieval and ICSI a possible treatment [Reference Westlander, Ekerhovd and Bergh9,Reference Rosenlund, Kvist and Ploen10].
Protection from the immune system is mediated by neighbouring Sertoli cells forming tight junctions between them, thereby dividing the seminiferous tubules into two compartments: (1) the basal compartment facing the ‘inside’ where the immune system can act against ‘foreign’ objects, and (2) the one facing the ‘outside world’ – the luminal compartment, i.e. the lumen into which the spermatozoa are released and transported out of the man. At meiosis, the primary spermatocytes, which soon are going to give rise to spermatids with unique DNA (and therefore will appear as ‘foreign’ to the immune system), are transferred from the basal to the luminal (outside) compartment, thus escaping the risk of being attacked by the immune system. The so-called blood-testis barrier consists of a combination of these inter-Sertoli cell connections, the peritubular tissue in the walls of the tubules, and the endothelium of the testicular capillaries in the interstitium between the tubules [Reference Holstein, Schulze and Davidoff7].
In response to hCG from the pregnant ovary or the placenta, Leydig cells produce testosterone necessary for the differentiation and development of male genitalia. Disturbed function of the ovary or placenta during critical time intervals during embryonic development can jeopardize the specific male development and hence future male fertility.
At puberty, GnRH is secreted from the hypothalamus in isolated peaks (one every 90 min), which stimulates the pituitary to secrete FSH and LH (named luteinizing hormone from its effect in females). LH stimulates the Leydig cells to produce testosterone. No less than 90% of the testosterone is taken up by the Sertoli cells in the tubules and is used to support spermatogenesis and by luminal flow for the androgen-specific functions of the excurrent duct system up to the corpus of the epididymis. Some 10% is delivered to the capillary blood and exerts systemic androgen effects on the man, including male secondary sex characteristics like body and facial hair, deep voice, increased muscle mass, decreased body fat, increased male haemoglobin, and enforced skeleton as well as brain function resulting in a ‘male temperament’ [Reference Nieschlag, Behre and Nieschlag6].
The Human Spermatozoon
The Messenger Cell
The morphology of the human spermatozoon is depicted in Figure 2.4, drawn by the German scientist Adolph Holstein, based on electron microscopic studies [Reference Holstein, Schulze and Davidoff7]. An extensive review of human sperm structure and function has recently been published by Mortimer [Reference Mortimer11]. The spermatozoon is a messenger cell – a conveyer of information – carrying the unique paternal messages needed to create a healthy child and grandchildren. As a messenger it needs special properties, such as being motile in order to reach the immotile oocyte and deliver its information after fusion with the oocyte membrane. The motility of the spermatozoon depends on the axoneme structures (e.g. microtubule doublets, dynein arms, spokes), the presence of functional mitochondria, functional centrioles (tail insertion), and fibrous tail sheath (rigid tail movements).
The Sperm Chromatin, DNA, Protamines and Zinc
The DNA of the spermatozoon is temporarily well-protected in a semi-crystalline structure stabilized by zinc (Figure 2.5). This unique, dense packaging of sperm chromatin offers protection and could compensate for the lack of DNA repair systems. The sperm chromatin is primarily arranged like a ‘rope’ with three intermingled strands: the two DNA strands constitute two strands while the third strand is constituted of protamines. There is one zinc ion for every protamine molecule and turn of the DNA helix, i.e. for every 10 base-pairs of DNA. Zinc withdrawal enables a quick unwrapping of the chromatin into separate DNA threads (double-stranded helices), when studied almost immediately after ejaculation. This ability promptly declines among spermatozoa left in the liquefying ejaculate in vitro [Reference Kvist1,Reference Kvist, Björndahl and Kjellberg12–Reference Björndahl and Kvist14].
This unique, dense packaging of the sperm genome is due to exchange of DNA-binding histones in the nucleus. During the late phases of spermiogenesis, somatic histones are replaced first by temporary proteins and then by protamines. At the same time, zinc is incorporated into the sperm nucleus. The positive amino groups (-NH3+) of arginine in the protamines neutralize the negative phosphate groups(-PO43-) of the DNA-backbone, which is the basis for the tight packing of the DNA-protamine complex [Reference Björndahl and Kvist15,Reference Björndahl and Kvist16].
This allows chromatin fibres to be closely aligned side by side and coiled into toroids with a diameter of 50–100 nm with 50 000 base pairs of DNA [Reference Björndahl and Kvist16–Reference Kvist, Afzelius and Nilsson19]. It appears that starting from the centromere, the p- and q-arms of the chromosomes separately form rows of piled toroids (see, for example, Figure 6 in ).
In human sperm chromatin there are mainly two types of protamines, protamine 1 and protamine 2. Besides arginine, these protamines contain the amino acids cysteine and histidine that can form stable temporary salt-bridges with zinc. The NH group of the imidazole ring of histidine and the reduced SH group of the cysteine have high affinity for zinc (c.f. zinc-fingers). Moreover, the presence of zinc facilitates the binding of DNA to protamine [Reference Björndahl and Kvist15,Reference Bal, Dyba and Szewczuk21,Reference Brewer, Corzett and Balhorn22]. Sperm nuclear zinc deficiency induced in vitro provokes liberated sulfhydryl groups to form disulfide-bridges and thereby changing the type of chromatin stability from zinc-dependent to non-zinc-dependent chromatin stability caused by disulphide-bridges leading to an abnormal (covalently bonded) and less easily reversible stabilization of the chromatin (Figure 2.6). This is likely to hinder or at least delay sperm chromatin decondensation in the ooplasm and could therefore be an important cause for male factor infertility.
Sperm Mitochondrial DNA and Impaired Spermatogenesis
Each mitochondrion in the body contains several copies of circular mitochondrial DNA (mtDNA). Although most of the genome is packaged into the chromosomes within the nucleus, mitochondria also have a small amount of their own DNA. In humans, mitochondrial DNA comprises 16,559 base pairs which constitute 37 genes, all of which are essential for normal mitochondrial function: 13 are for enzymes involved in oxidative phosphorylation (OXPHOS), while the remaining 24 code for transfer and ribosomal RNAs (i.e. tRNA and rRNA molecules). The OXPHOS enzymes are not coded for within the nuclear genome of mammalian cells.
At fertilization, the spermatozoon brings some 100 copies of mitochondrial DNA, whereas the oocyte has some 100,000 copies. Sperm mtDNA has passed up to 400 cell divisions and is more likely to be mutated than the mtDNA in the oocyte that have been selected, multiplied and then kept resting since the tenth fetal week. Sperm mitochondrial DNA is ubiquitin-targeted already in the testis to be destroyed upon entrance into the oocyte [Reference Sutovsky and Song23,Reference Birkhead and Immler24]. Thus, mitochondria are inherited through the female germ cell line. There are reports suggesting that mutated sperm mitochondrial DNA may escape destruction and cause mitochondrial disease in the offspring. If so, it seems to be an extremely rare event.
Mutagenic damage to mitochondrial DNA (mtDNA) is a condition that limits the lifespan of each individual. By regeneration of mitochondria with undamaged native mtDNA in the oocyte, a new individual is provided with fully functional mitochondria from the one cell stage or zygote. This appears to be the evolutionary mechanism for family eternity, by which every new generation can start off with fresh mitochondria.
Mutagenic deletions of mitochondrial DNA are likely to propagate relatively rapidly in cells with frequent cell divisions. For a 35-year-old man, his spermatogonia have undergone some 400 mitotic divisions. This means that a deletion of the mtDNA in a spermatogonium would lead to a metabolic exhaustion of the processes involved in spermatogenesis and could therefore be manifested by, for example, severely reduced sperm concentration, motility and morphology. Thus, genetic characterization of germ cell mtDNA or sperm mtDNA could be a future tool to estimate whether disorders in spermatogenesis are due to inadequate mitochondrial functions.
Spermatogenesis in man appears to be a process of redundancy. Developing germ cells and spermatozoa are lost during and after spermatogenesis, and only some 25% of spermatozoa formed reach the ejaculate [Reference Holstein, Schulze and Davidoff7]. Among them, the proportion of malformed spermatozoa is extremely high. One can argue that if the man has at least some morphologically ‘ideal’ (previously referred to as ‘morphologically normal’) spermatozoa (i.e. the structure and form of the spermatozoa makes them able to pass through cervical mucus and bind to the zona pellucida), then the genetic ‘blueprints’ for a typical spermatozoon are present. But, control of the process is apparently of very low priority for fertility, and is therefore not given high efficiency in terms of biological quality management. This type of pleiomorphism (large range of sperm forms in the ejaculate) is almost never seen in laboratory animals or inbred farming animals, where spermatozoa have excellent morphology. This may be due to natural selection when males compete for the oocytes at the sperm level, or human selection of animals with good sperm morphology. Besides humans, gorillas and some monogamous mouse strains have pleiomorphic spermatozoa. A plausible explanation for this could be that the performance (and thereby the structure and function) of spermatozoa is not critical when the competition for reproduction is between males (the female only receives spermatozoa from one male), rather than between their spermatozoa (the female receive spermatozoa from several males during an ovulatory cycle) [Reference Birkhead and Immler24]. It should be emphasized that if a man has only 1% of spermatozoa with morphology typical for those spermatozoa that reach the site for fertilization, an ejaculate of 100 million spermatozoa still contains 1 million spermatozoa with ‘ideal’ morphology.
Note that pleiomorphism means that many different types of morphological variants exist in an ejaculate. This must be distinguished from conditions where most spermatozoa have the same type of atypical morphology. In such cases a genetic reason might be suspected and a chromosomal analysis (karyotype) can rule out chromosomal translocations and inversions. In fact, sperm morphology screening in mice is used as a method to monitor toxicological exposures that induce chromosomal translocations.
Sperm Transport: From Testis to Urethra
Testicular spermatozoa are transported from the seminiferous tubules in the testis by the flow of fluid to the rete testis and then by the 15–20 efferent ducts to the convoluted, approximately 6 m long, epididymal duct on each side (Figure 2.7). The epididymis is anatomically divided into the caput, corpus and cauda regions (Figure 2.8). There is also a more functional subdivision of the epididymal duct into the initial, middle and terminal segments. The epididymal duct, and its continuation into the vas deferens and the seminal vesicles, is developed from the embryonic Wolffian duct. In the vas deferens, the spermatozoa are transported from the distal cauda to the urethra. Before passing through the prostate, the vas deferens widens to form the ampulla of the vas deferens, from which the seminal vesicles develop. The ampulla and the seminal vesicle on each side have a common excurrent duct named the ejaculatory duct, which opens into the urethra [Reference Neill5,Reference Nieschlag, Behre and Nieschlag6].
The differentiation, development and secretory function of these organs are dependent on androgens. From the testes to corpus of the epididymis, most androgens are provided by the local fluid transportation from the testis (luminal fluid, lymphatic fluid, and local venous plexa), while the systemic circulation provides androgens to the cauda, vas deferens, seminal vesicles and prostate. The prostate has developed from the embryonic genitourinary sinus.
In most mammals the epididymal transit time has been reported to be 7–10 days. However, the transit time is dependent on the amount of spermatozoa to be transported (i.e. daily sperm production). Men with high sperm output (>200 × 106 spermatozoa per day) had an average transit time of two days, whereas men with lower output (mean approximately 70 × 106 per day) reached six days of transit time [Reference Johnson and Varner25]. The luminal transport from corpus to cauda seems independent of nervous regulation but involves local spontaneous waves of contraction, 7–9 per min. In the distal cauda and vas deferens, the spontaneous contractility is only 1–2 waves per min, resulting in an accumulation of spermatozoa in the distal cauda (i.e. it is a region of sperm storage).
Transit time through the caput, corpus and proximal cauda is independent of ejaculatory frequency. The transport of spermatozoa from the distal cauda to the urethra is dependent on neurogenic activity, and frequent ejaculation results in a decline in sperm numbers there, while sexual abstinence results in increased numbers stored. One ejaculation a day for two days seems enough to normalize and equalize an increased or depleted sperm storage to the level of the daily sperm output [Reference Johnson26].
During epididymal transit, the spermatozoon undergoes a maturation process by which it acquires capabilities as a messenger cell to traverse and survive the female genital tract and eventually deliver its genetic information to the embryo-to-be. Among these properties are progressive motility, the ability to fuse with and fertilize the oocyte, and the ability to sustain embryonic development into a viable offspring. The maturation process appears not to be restricted to particular parts of the epididymis but occurs as a function of time. Thus, spermatozoa withheld in the caput by occlusion of the duct in the corpus develop fertilizing capacity. Normal development and function of the caput and corpus epididymides is dependent of luminal flow of testosterone, which is locally transformed to the active dihydrotestosterone by the converting enzyme 5-α-reductase type 1.
The cauda epididymidis has evolved as the sperm storage organ in animals forced to wait for female ovulation. Is seems that when reaching the proximal cauda, the spermatozoa have achieved ‘conservation factors’ enabling prolonged storage. Two major factors contributing to this sperm storage are: (1) a low temperature (34°C) and (2) an androgen-dependent environment, including secretory products, created in the cauda epididymidis. The androgen acting in the cauda epididymidis is testosterone, transported by the systemic circulation and taken up by the epididymal epithelial cells and locally converted to dihydrotestosterone by 5-α-reductase type 2, (5-αR2). Factors jeopardizing the scrotal temperature and the testosterone effects (e.g. compounds interacting with 5-αR2) would decrease the functional storage time.
The conservation processes involve inactivation of motility and metabolism, and the stabilization of various sperm structures and membranes. At ejaculation, the mixing of spermatozoa with the prostatic fluid restores motility, and it is plausible that the capacitation process needed to activate mammalian spermatozoa for fertilization involves restoration of conserved mechanisms [Reference Bedford27].
The Size of the Sperm Storage
As a species, humans exhibit relatively low sperm production, a mere 100–500 × 106 per day. Sperm production is continuous and, like the ram, a man can have several ejaculations a day with fertile spermatozoa. Ejaculation every fourth hour after three days of abstinence can give results like 1000 × 106 in the first ejaculation, 125 × 106 just 4 h later, and 20 × 106 another 4 h later. It is worth noting that a decline from 1000 × 106 to 20 × 106 fresh spermatozoa due to sexual activity has no negative impact on the man’s fertility. In this respect the evaluation of sperm number is totally different from the evaluation of red and white cells in blood. Therefore, when evaluating sperm counts, the ejaculatory frequency (i.e. sexual behaviour) is of critical importance: not only the time interval between the preceding ejaculation to that collected for investigation, but also the frequency of ejaculations preceding collection for investigation. Of all the spermatozoa in the epididymis, 50–80% are localized in the cauda epididymidis, and half of them are available for ejaculation.
Experiments show that spermatozoa aged within the epididymis (or in the laboratory or within the female genital tract) first lose their potential to contribute to a normal embryonic development (probably due to the fact that prolonged sperm storage results in sperm DNA strand breaks and chromosomal aberrations of the embryos [Reference Björndahl and Kvist15]). Thereafter, they lose the ability to fully decondense their nuclear chromatin within the oocyte; followed by the ability to fertilize the oocyte and, long thereafter, they have a reduction in motility.Animal studies have shown that if the transport of fresh spermatozoa from the testis to the cauda epididymidis is hindered, those spermatozoa remaining in the cauda more frequently cause aneuploidies in resulting embryos after only six days in the epididymis. The question arises, therefore, how those spermatozoa that upon aging in the epididymis have damaged DNA, are eliminated and hindered in reaching the site of fertilization. Possible explanations include:
1) That old spermatozoa are mixed with fresh ones in the cauda epididymidis, and these fresh gametes with undamaged DNA are more likely to reach the oocyte. There is so far no evidence that the female genital tract can select spermatozoa based upon their genomes, and about 0.5–1% of newborns have chromosomal aberrations. Moreover, DNA damage occurs before the ability to fertilize is decreased.
2) Male sexual drive could result in nocturnal emissions, masturbation and non-ovulation-related intercourse, which in turn would eliminate old spermatozoa, making way for fresh ones.
3) Local muscle contractions of the cauda epididymidis/vas deferens can emit spermatozoa to the urethra where they are voided with the urine. Spermatozoa in morning urine is diagnostic for spermarche in boys [Reference Johnson and Varner25,Reference Richardson and Short28].
The vas deferens can be palpated as a 3–5 mm thick ‘string’ in both sides of the scrotum. The vas has three robust layers of smooth muscle, one outer longitudinal, one circular and one inner longitudinal, and facing the lumen is a convoluted mucosal layer. The epithelium has one layer with secretory cells. Stimulation of sympathetic neurons releases noradrenalin which stimulates adrenergic α1-receptors on the smooth muscles leading to a mass-contraction that, within a second, transports spermatozoa from the distal cauda epididymidis to the urethra.
The seminal vesicles and the ampullae have one layer of secretory epithelium of common origin that is highly convoluted. The secretory cells are stimulated by sympathetic neurons using acetylcholine as the transmitter; acetylcholine stimulates the formation of androgen-specific secretory products like fructose. The smooth muscles of the walls are stimulated to contract by the sympathetic adrenergic neurons; noradrenalin stimulates adrenergic α1-receptors which initiates contraction whereby the contents are emitted to the urethra. The ejaculatory ducts open in the prostatic part of the urethra. In cases of agenesis of the Wolffian duct system (epididymides, vasa deferentia, seminal vesicles and ejaculatory ducts), either parts of the system or the whole system is missing. The ejaculate then lacks secretory markers for the missing parts (neutral α-glucosidase for the epididymis, fructose for the seminal vesicles and ampullae).
The Physiological Role of the Seminal Vesicles in Humans Is Unknown
Human spermatozoa ejaculated in, or incubated in, seminal vesicular fluid show decreased motility, vitality, decreased chromatin zinc content, and profound changes in chromatin stability.
In many textbooks, fructose is mentioned as a substrate for sperm metabolism. However, considering the negative effects that seminal vesicular fluid has on spermatozoa (decrease in vitality, motility and affected chromatin packaging), and that in vivo spermatozoa normally do not come into contact with seminal vesicular fluid, the paradigm of seminal fructose being a substrate for human spermatozoa should be challenged [Reference Mann and Lutwak-Mann29]. Another role for fructose, and other ‘unusual’ sugar-types in semen of other animals could be to normalize the osmotic pressure to 290 mOsm/l. The seminal vesicles contain 40 million times higher concentrations of prostaglandins than the blood, and their physiological role also remains to be clearly determined.
The prostate is composed of 20 to 30 different glandular acini that open into the urethra. These glands evolved as branching buds from the sinus urogenitalis (i.e. an origin similar to the lower parts of the vagina). Testosterone from the systemic circulation is locally transformed to 5-α dihydro-testosterone by 5-αR2. Sympathetic cholinergic nerves stimulate the formation of androgen-specific secretory products as zinc, magnesium, calcium, citrate, acid phosphatase, and prostate specific antigen (PSA) [Reference Mann and Lutwak-Mann29]. Anti-cholinergic compounds thus counteract the formation of prostatic secretion, as do inhibitors of testosterone-dehydrogenase type 2. At emission, smooth muscle cells surrounding the glands are contracted and the fluid from the 20 to 30 glands is expelled and mixed with spermatozoa in the urethra. Emission is mediated by the adrenergic α1-receptors on the smooth muscle cells stimulated by noradrenalin released from sympathetic adrenergic neurons.
Prostatic fluid also contains many substances that are normally present in blood plasma and are transudated from the blood plasma to the prostatic fluid. An acute inflammatory reaction in the prostate increases the transudated part, resulting in higher semen volume with lower concentration of androgen-specific compounds.
Emission involves emptying of spermatozoa and the various fluids into the urethra. The sympathetic neurons also release noradrenalin on the smooth muscles surrounding the urinary bladder neck, which results in a closure of the urethra, preventing the emitted spermatozoa and fluid from passing up into the urinary bladder.
Diseases or surgery affecting the sympathetic neurons, e.g. diabetes, transurethral resection of the prostate (TUR) or retro-peritoneal-lymph-node-dissection (RPLND), can result in disturbed emission. Either there is no, or decreased, emptying of spermatozoa and fluid into the urethra, or fluid is expelled up into the urinary bladder. This condition results in lower or no volume expelled at ejaculation, with little or no antegrade ejaculation. Presence of large numbers of spermatozoa in urine collected after orgasm means that there has been a retrograde ejaculation.
Often, low or no antegrade ejaculation is associated with impaired emission from the epididymis and the glands, and no or few spermatozoa are found in the urine. In some cases, α1-agonists can stimulate emission, bladder neck closure and result in antegrade ejaculation.
The ductuli efferentes, the Wolffian duct system, the prostate and the bladder neck constitute a functional transport system of smooth muscles. In the efferent ducts, caput, corpus and proximal cauda epididymides, the smooth muscles are rich in intercellular tight junctions. The consequence is that spontaneous electrical depolarizations can be spread and induce waves of spontaneous contractions transporting spermatozoa and fluids without regulation by nerves. In contrast, the smooth muscles of the distal cauda, the vas deferens, the seminal vesicles, the ejaculatory ducts, the prostate and the bladder neck, have fewer junctions and are therefore dependent on neurogenic stimulation to induce waves of contraction [Reference Wagner, Sjöstrand and Sjösten31].
The post-synaptic nerves stimulating the smooth muscles are specific to the genital tract and are called short adrenergic neurons. The pre-synaptic sympathetic neurons emanate from the lateral horns of the thoracic and lumbar regions of the spinal cord. They reach the genitalia through the hypogastric plexus and the hypogastric nerves running lateral to the rectum. Inside the genital organs they are connected to the post-synaptic short neurons. Ejaculation induced by vibrators uses, as do masturbation or coitus, the whole emission reflex. Electric stimulation of the hypogastric plexus or hypogastric nerves result in contractions in the distal cauda, vas deferens, the seminal vesicles, the ejaculatory ducts, the prostate and the bladder neck. By rectal electro-stimulation, various parts of the system can be activated to cause emission without the normal ejaculatory sequence.
The smooth muscles in the whole system respond to noradrenergic α1-stimulation. Emission can be partially or completely blocked by α1-receptor blockers (e.g. phentolamine and some anti-depressive agents) and is augmented by α1-agonists (e.g. phenylpropanolamine, as used against oedema in the nasal mucosa). The adrenergic neurons are inhibited by the cholinergic neurons that stimulate secretion. Thus, during formation of fluid during sexual arousal, emission can be partly inhibited.
The secretory cells in the epididymis, the vas deferens, the ampulla, the seminal vesicles and the prostate are innervated by short sympathetic cholinergic neurons releasing acetylcholine. The secretory neurons are inhibited by the adrenergic neurons. Thus, secretory stimulation decreases upon emission.
At orgasm, spermatozoa in the cauda of the epididymis are emitted into the urethra and suspended in prostatic fluid. Striated muscles in the pelvis contract and increase the pressure in the urethra so that its contents are expelled. Later expelled fractions are mainly composed of a fluid from the seminal vesicles, which form a gel. Spermatozoa in the zinc-rich prostatic fluid preserve motility, vitality and a zinc-dependent stabilization of their nuclear chromatin. Spermatozoa that meet seminal vesicular fluid lose motility, vitality and zinc and the normal stabilization of the nuclear chromatin [Reference Kvist1,Reference Björndahl and Kvist14].
The urethral walls are extended by prostatic fluid with spermatozoa. This extension evokes somatic reflexes of rhythmic contractions in the bulbo- and ischio-cavernosus muscles and also in the muscles of the pelvic floor. The bulbocavernosus muscle surrounds the corpus spongiosum of the penis and inserts into the corpus cavernosum of both sides. The ischio-cavernosus muscles insert into the crus of the penis on both sides. These striated muscles of the perineum are innervated by the pudendal nerve. Note that whereas emission is controlled by autonomic nerves, ejaculation is governed by striated muscles under ‘voluntary’ control. However, men seldom practice to train these muscles, as done with the striated muscles controlling the bladder and the rectum, so ejaculation is experienced as an involuntary process once orgasm is reached.
The contractions create an increase in intra-urethral pressure which forces the urethral contents outwards through the penis (when the bladder neck is closed). The flow speed of the ejaculate portions is dependent on how effectively the contraction force is transduced into increased pressure in the urethra lumen. The more powerful the erection the more of the force is transferred to pressure, giving a high flow velocity and a good separation of the ejaculate fractions. In contrast, a poor erection means more penile plasticity and less separation of ejaculatory fractions.
The high ejaculatory flow-speed is physiological, but also one reason why the sperm-containing first fraction of an ejaculate is often lost at semen collection by masturbation. Such ejaculates, where many (most?) of the spermatozoa have escaped collection, should not, of course, be used to evaluate sperm production.
Our knowledge of the normal ejaculatory sequence comes from studies using split-ejaculates [Reference Björndahl and Kvist14,Reference Mann and Lutwak-Mann29,Reference Amelar and Hotchkiss32]. At ejaculation, each fraction was collected and characterized by counting the spermatozoa and measuring secretory markers for the prostate (zinc) and the seminal vesicles (fructose). Such studies revealed that spermatozoa from the distal cauda epididymidis are suspended in the simultaneously emitted fluid from the prostatic glands and expelled in a first ejaculatory portion: thus, the physiological ejaculate that is deposited onto the cervical mucus comprises spermatozoa in prostatic fluid. Shortly thereafter, fluid from the seminal vesicles is expelled. A specific sequence of ejaculation is also true for several animals; for example, in the boar, where the sperm-rich fraction is collected for assisted reproduction.
Abnormal Ejaculatory Sequence
A common, but often neglected, disorder is delayed emptying of the prostatic fluid, seen especially in men with inflammatory reaction of the prostate [Reference Amelar and Hotchkiss32–Reference Kvist34]. The spermatozoa are then primarily expelled mixed with the seminal vesicular fluid, which has the potential to affect sperm motility, vitality and chromatin packaging. Amelar and Hotchkiss noted that some 6% of infertile men had a delayed expulsion of spermatozoa and that the sperm-rich fraction of the ejaculate was more successful for insemination [Reference Amelar and Hotchkiss32].
Redistribution of Zinc at Ejaculation
Zinc is secreted as both free ions and ions bound to citrate in the prostatic fluid. Seminal vesicular fluid contains powerful zinc-binding, high-molecular weight proteins which can extract zinc from spermatozoa and redistribute the citrate-bound and free zinc to the proteins of vesicular origin during and after liquefaction [Reference Björndahl, Kjellberg and Kvist33–Reference Arver36]. The proportion of zinc bound to high molecular weight proteins shows huge variations between men and ejaculates (2–67%) [Reference Kjellberg, Björndahl and Kvist37]. Although a semen sample might have high zinc concentration, compounds in the liquefied ejaculate can act as zinc chelators, thereby adversely affecting the sperm chromatin.
Forces that normally stabilize the sperm chromatin seem essential for a safe transfer of the genetic material to the oocyte. Fertile sperm donors have been shown to have higher sperm chromatin zinc content than infertile men, and some 25% of infertile men can have altered chromatin stability. The chance for pregnancy at IVF is severely reduced among men with low seminal zinc concentration. Zinc withdrawal enables a quick unwrapping of the whole nucleus into separate threads, when studied within minutes after ejaculation. This ability promptly declines when spermatozoa are stored in vitro [Reference Kvist, Björndahl and Kjellberg12,Reference Björndahl and Kvist15]. Compounds from the seminal vesicles can deprive the sperm chromatin of zinc, affecting the normal protective packaging of the sperm DNA. It should be recalled that in vivo the spermatozoa are expelled in the zinc-rich prostatic fluid and enter the cervical mucus immediately at ejaculation without exposure to zinc-depriving seminal vesicular fluid.
Recently, it was reported that ejaculates with a high proportion of spermatozoa that had acquired chromatin zinc deficiency during or after liquefaction were the ejaculates that contained high proportions of spermatozoa with fragmentated sperm DNA [Reference Houska, Flanagan, Björndahl and Kvist38,Reference Kvist, Flanagan, Björndahl and Kvist39]. Hence it appears that the unphysiological way in which we collect semen and expose spermatozoa to seminal vesicular fluid during liquefaction can cause semen samples for use in ART to undergo oxidative sperm DNA damage.
In reality, the ‘standardized’ semen sample only exists in the laboratory, when the entire ejaculate is collected and mixed in a single container. From a physiological perspective, the ‘standard semen sample’ is therefore an artefact that forces spermatozoa in the prostatic fluid to be trapped by the expanding vesicular gel for up to 20 min. Prostate Specific Antigen (PSA) from the prostate degrades the gel-forming seminogelins of vesicular origin. Due to this process, and to the general conditions during the in vitro handling procedures, the spermatozoa are exposed to increasing osmolarity, increased oxygen tension and elevated levels of free radicals [Reference Kvist1,Reference Björndahl and Kvist14].
In vivo, spermatozoa are expelled in the isotonic prostatic fluid onto the isotonic cervical mucus extruding from the cervical opening into the vagina. In vitro, enzymatic digestion of macromolecules starts at ejaculation, resulting in a rapid increase of the semen osmolarity from 290 up to 450 mOsmol/l by 3 h later [Reference Björndahl and Kvist14,Reference Kvist, Flanagan, Björndahl and Kvist39–Reference Holmes, Björndahl and Kvist42].
There are two implications of this: (1) the possible direct effect of the increasing osmolarity on spermatozoa; and (2) the risk and effects of hypo-osmotic stress, when spermatozoa in hypertonic seminal plasma later are exposed to isotonic culture media used for sperm selection.
Increasing osmolarity in the liquefied ejaculate: The actual osmolarity reached and its rate of increase vary between samples [Reference Holmes, Bjorndahl and Kvist41]. Increased semen osmolarity is strongly associated with decreased sperm motility [Reference Holmes, Bjorndahl and Kvist41]. When semen osmolarity increases the spermatozoon loses water, which activates volume-regulating mechanisms to restore the cellular volume, a process that requires energy. Continuously increasing osmolarity evokes continuous volume regulation that could exhaust the spermatozoa by consuming their intracellular ATP, which is also needed for other cellular functions such as motility. Ejaculates incubated at higher osmolality therefore show reduced sperm motility and velocity [Reference Holmes, Flanagan, Björndahl and Kvist43], and semen samples with impaired motility had higher osmolality than controls [Reference Makler, David, Blumenfeld and Better44–Reference Velazquez, Pedron, Delgado and Rosado46].
Abruptly decreased osmolarity – the unintended hypo-osmotic shock at sperm selection: This hypo-osmotic challenge occurs when the spermatozoon that had adjusted to increasing osmolality abruptly are exposed to isotonic (~290 mOsm/l) selection media. After semen liquefaction and before sperm selection osmolarity levels increase from 290mOsm/l at ejaculation to 330–350–380 mOsm/l. Thus, the hypo-osmotic shock varies conspicuously between various samples ranging from 40–90 mOsm/l [Reference Holmes, Bjorndahl and Kvist41].
Living spermatozoa with intact membranes take up water and swell, an almost instantaneous effect described by Kölliker in 1856 . The size of the osmotic shock is the main factor that determines the amount of water to be taken up. The amount of water taken up determines the degree of swelling response seen in the tail, causing a variable degree of distal tail coiling and tail folding within its plasma membrane: tail tip coiling being the mildest effect. Up to now this iatrogenic osmotic shock has unintendedly been imposed on the spermatozoon in most samples used for ART.
In contrast to the inadvertent osmotic shock, the osmotic difference that spermatozoa are exposed to in the hypo-osmotic swelling (HOS) test is fully intended. The HOS test was developed to reveal which spermatozoa are alive and able to take up water, swell and coil their tails in response to exposure to an osmolarity of 175 mOsm/l [Reference Jeyendran, Van der Ven and Zaneveld47], i.e. an osmotic shock of 155–205 mOsm/l (which is far greater than the 40–90 mOsm/l shock that occurs during sperm selection for ART.
The HOS test will cause excessive water uptake and results in more advanced sperm swelling and many spermatozoa react with full coiling of the tail that can be seen either as a circular structure at the base of the head or as total coiling of the tail around the sperm head.
Since the osmotic shock induced at sperm selection seldom results in totally coiled tails, the consequences of exposing spermatozoa in the ejaculate to ‘isotonic’ selection media has unfortunately remained unrecognized until recently [Reference Holmes, Flanagan, Björndahl and Kvist43]. It was observed that ejaculated spermatozoa exposed to isotonic selection media swam in a jerky fashion. Freeze-frame video recordings of jerky swimming spermatozoa revealed spermatozoa with morphological deformed tails that were coiled at the tips or partly folded, irreversible morphological changes that did not change within an hour [Reference Holmes, Flanagan, Björndahl and Kvist43].
One approach to eliminate the effects of increasing osmolarity would be to apply the selection provided by nature, i.e. to select spermatozoa at ejaculation, for instance by using the first, non-coagulated split ejaculate fraction for density gradient centrifugation within minutes after ejaculation, or by early dilution of the ejaculate [Reference Björndahl and Kvist14,Reference Holmes, Flanagan, Björndahl and Kvist43].
Just 10–200 spermatozoa normally reach the ampullar parts of the oviducts; of which only one will fertilize [Reference Ahlgren48,Reference Mortimer and Templeton49]. Fatherhood has been proven among vasectomized men without any detectable spermatozoa in the ejaculate. Finding no spermatozoa in one 10 µm deep chamber only tells us that the probability is 95% that the sample has less than 720,000 spermatozoa. No spermatozoa observed in a centrifuged sample after examination of microscope 400 fields tells us, with the same probability, that there are less than 200 spermatozoa in the sample. Thus, if no spermatozoa are found under the microscope it does not exclude the possibility that there were spermatozoa present in the ejaculate.
In a study from Norway, men contributing to pregnancy within one month had higher total sperm number (mean 410 × 106 per ejaculate) compared to men needing up to 12 months to contribute to a pregnancy (mean 254 × 106 per ejaculate). Of all men contributing to a pregnancy within one year, 95% had ≥22 × 106 sperm per ejaculate [Reference Haugen, Egeland and Magnus50]. Among infertile couples in France, studied after a period of at least six months of infertility, those with <5 × 106 spermatozoa per ml, i.e. approximately corresponding to a total number of 22 × 106 per ejaculate in the study above, had a low (11%) chance of achieving a pregnancy within the next year whereas men with >5 × 106/ml had a 62% probability of contributing to a pregnancy during the same time [Reference Jouannet, Ducot, Feneux and Spira51]. A cut-off of 5 × 106/ml was also found in a Danish study [Reference Bostofte, Serup and Rebbe52], while another Danish study advocated a cut-off concentration of 40 × 106 per ml (corresponding to ~170 × 106 spermatozoa per ejaculate). However, the confidence interval for this chosen level was zero to infinity, and the data given allowed the reader to recalculate a cut-off level close to 10 × 106/ml. Moreover, some 20% of the men included in this study failed to collect the first, sperm-rich fraction, biasing the results and conclusions [Reference Bonde, Ernst and Jensen53]. Thus, a clinical cut-off for the chance to achieve pregnancy within one year seems close to 20 × 106 spermatozoa per ejaculate.
Studies of the integrity of sperm DNA show that men with few spermatozoa have spermatozoa with more chromosomal aberrations and more DNA strand breaks.
The spermatozoon is the only cell in the body that lacks DNA repair systems. This means that damage to the DNA during sperm formation, maturation, storage, ejaculation, in vitro handling, and transfer to the oocyte cannot be repaired by the spermatozoon itself. DNA damage does occur and can be repaired by the oocyte, although this repair could be complete, wrong or incomplete. Faulty repair might result in de novo translocations (chromosome strand sections containing genes that are wrongly transferred onto other chromosomes) or inversions (chromosome strand sections containing genes that are inserted onto the right chromosome but in the opposite direction). If all the genetic material is still present, the translocation or inversion is called balanced, but if not, it is unbalanced. If DNA strand breaks are left unrepaired, genes can be lost and the condition is called a chromosome deletion. Deletions and unbalanced aberrations often result in miscarriage or malformations, and can also affect psychomotor development after birth. A balanced translocation results in a healthy child with normal psychomotor development and normal puberty. However, its own fertility could be reduced and it has higher risk of contributing to miscarriage, fetal death, malformations and affected psychomotor development in the subsequent generation. This is because individuals with balanced translocation produce gametes with variable genomes: some gametes have too much DNA, some too little, some have normal DNA, and some have the same balanced translocation as the individual himself.
A spermatozoon used for fertilization that has an acquired DNA damage might therefore not only affect the child-to-be but also, or only, affect the generation thereafter. Consequently, the full safety of any assisted reproduction method can only be judged using a two-generation perspective [Reference Kvist1,Reference Aitken and Bakos54–Reference Vaughan, Tirado and Garcia56].
With the evolution from external to internal fertilization, and internal embryonic and fetal development, the numbers of embryos and fetuses must be limited. Females who ovulated multiple oocytes were at an evolutionary disadvantage. Males are, in this evolutionary aspect, still at primitive invertebrate level, i.e. 100 million genetic lots are produced each day and released in vast numbers. Human females are anatomically unsuited for multiple pregnancies. The increased prevalence of obstetrical problems and adverse outcomes in spontaneous twin pregnancies are eloquent expressions of humans being an essentially monotocous species
However, in some 1% of families there is a hereditary propensity for spontaneous twin pregnancies. In these cases, the survival of females and fetuses has been less incompatible with twin pregnancies. This is, however, not the case for the twin pregnancies produced by ART [Reference Kvist1].
The evolutionary pressure on the female side that resulted in the ovulation of a single oocyte created another problem for nature. The single oocyte is threatened by an invasion, 100 × 106 spermatozoa against one single oocyte, and evolutionary biologists discuss the need for the female to protect her oocyte from this invasion. The cervical mucus, the isthmus of the Fallopian tubes, and the barriers protecting the oocyte can be viewed as central components of this defence.
From a physiological point of view the cervical mucus is where the male deposits the first expelled population of spermatozoa suspended in prostatic fluid (Figure 2.9). Thereafter, the gel-forming seminal vesicular fluid is expelled – and there is no evidence that these two secretions are mixed in the vagina as they are mixed in a semen collection vessel. Rather, it seems that in vivo the freely swimming spermatozoa in the prostatic fluid, some of which enter the cervical mucus, and the gel-formed vesicular secretion form two separate compartments. In many other mammalian species, the seminal vesicular secretion forms a firm copulatory plug that prevents other males from mating successfully with the female, so in this respect the human seminal vesicular contribution can be seen as being vestigial.
The cervix, its mostly impermeable mucus, and the low pH of the vagina, which is created by the vaginal microbiome [Reference Hong, Ma and Yin57,Reference Tsonis, Gkrozou and Paschopoulos58], are barriers in the female between micro-organisms in the outside world and the interior of the female reproductive tract and the peritoneal compartment.
To enable fertilization, these barriers must allow penetration of motile male gametes at ovulation. FSH leading to follicular development also results in increased levels of estradiol (E2) which stimulate the cervical glands to produce mucus that (1) does not kill the spermatozoa and (2) allows them to penetrate using their own power (motility). At ovulation, the cervical mucus has conditions optimal for sperm passage, the highest anti-bacterial activity and, at the same time, the pH of the vagina is the lowest. Soon after ovulation, increased levels of progesterone affect the cervical glands and make the mucus they secrete impenetrable to spermatozoa.
The textbook picture of the vagina as an open tube with cervical mucus restricted to the cervical canal must also be challenged. Close to ovulation the narrow, collapsed lumen of the vagina contains the ‘sperm-catching’ cervical mucus protruding from the external cervical os into the vagina – unless it has been interfered with by, for instance, hygiene procedures. Using natural family planning procedures, samples of cervical mucus are collected at the vulva. Clinical protocols for vaginal examination prescribe that this expression of physiology – protruding cervical mucus – should be removed to give the examiner a clear view of the cervix. However, the procedure can give the examiner the wrong impression that cervical mucus is restricted to the cervix only.
It has been suggested that the cervix functions as a sperm reservoir. However, while spermatozoa might have been found to survive deep up in the crypts of cervical mucus, this does not prove that spermatozoa temporarily surviving in the crypts actually constitute a reservoir since there is no evidence that these spermatozoa ever re-emerge from the crypts into the cervical canal [Reference Mortimer and Templeton49,Reference Suarez and Pacey59,Reference Mortimer, Grudzinskas and Yovich60].
The progressive motility of the spermatozoon is essential for it to penetrate into and pass through cervical mucus, and hence spermatozoa with poor motility are excluded or filtered out during their passage through it. Concomitantly, spermatozoa with abnormal morphology are also removed , resulting in only a minority of ejaculated spermatozoa actually entering the cervix [Reference Mortimer and Templeton49,Reference Suarez and Pacey59,Reference Mortimer, Grudzinskas and Yovich60]. Some hours after ejaculation, the uterus is invaded by leucocytes that attack the spermatozoa. It is therefore reasonable to assume that the fertilizing spermatozoon has to traverse the uterus and reach the immune-privileged oviductal epithelium in the isthmus of the oviduct ahead of this elimination of spermatozoa in the uterus. Uterine muscular contractions might enhance passage of spermatozoa through the uterine cavity to the utero-tubal junctions since radio-labelled spheres placed in the vagina can be translocated to the oviductal isthmus during the late follicular phase [Reference Suarez and Pacey59].
The anatomy of the utero-tubal junction, the folded mucosa and contracted smooth muscle layer, along with a mucus secretion that flows towards the uterus, together constitute a functional barrier for the spermatozoa to pass, as well as a barrier that can prevent a fertilized oocyte reaching the uterine cavity too soon. Short adrenergic neurons release noradrenalin that triggers the smooth muscles of the isthmus to contract when dominated by estradiol during the follicular phase before ovulation. Following ovulation, increased levels of progesterone hyperpolarize the smooth muscles, thus counteracting the adrenergic action and the muscles relax, allowing the zygote to pass at Day 3 to 4 after fertilization. There is some evidence indicating that more than good sperm morphology and progressive motility is needed to pass into the isthmus of the Fallopian tubes. A few thousand spermatozoa may swim through the utero-tubal junctions to reach the isthmus region of the oviducts. Whereas other mammals reveal a distinct sperm reservoir, the human isthmus constitutes at least a functional reservoir that could prolong the availability of spermatozoa maintained in a fertile state, by interacting with the oviductal epithelium. The perceived difference might be due to the fact that the proximal isthmus, which is the likely site of the sperm reservoir within the human female reproductive tract, is embedded within the thick muscular wall of the uterus, and therefore difficult to access for observational studies. Subpopulations of small numbers of spermatozoa become capacitated and hyperactivated, which enables them to proceed towards the tubal ampulla [Reference Mortimer61]. At any time only some 10–200 spermatozoa could be flushed from normal human oviducts, while some thousands could be recovered in distally obstructed oviducts [Reference Mortimer and Templeton49,Reference Suarez and Pacey59,Reference Mortimer, Grudzinskas and Yovich60].
Spermatozoa that reach the oviducts before the uterine invasion defence system reacts and impedes later arriving spermatozoa?
A subpopulation of spermatozoa that reaches the isthmus and has a special additional competence to pass?
A subpopulation that is invisible to the uterine invasion defence system?
If it is the first two groups, then spermatozoa in the first expelled ejaculate fraction would be more representative for these than the spermatozoa residing in the liquefied whole ejaculate. But if it is the latter two groups, then these spermatozoa would be true subpopulations that we need to be able to identify. In the future we might be able to identify and select spermatozoa that can reach the oviducts from within the first expelled fraction. But we can never truly predict whether a man will become a father or not, regardless of the sophistication of methods we use, as long as we only assess the ‘messenger’ functions of the spermatozoa. Also, the messages could only be studied in a number of spermatozoa representative of the selected subpopulations, since it is doubtful that we will ever be able to study the messages of a single spermatozoon without affecting its integrity [Reference Kvist1].
There is no valid support for a rapid (minutes) sperm transport, although some early observations indicated that human and rabbit spermatozoa could reach the ampulla minutes after insemination – although these spermatozoa were mostly dead and did not contribute to fertilization [Reference Suarez and Pacey59,Reference Mortimer, Grudzinskas and Yovich60]. Experimental studies to clarify the physiological situation, especially in humans, are lacking, and will be extremely difficult to perform.
Allowing speculation, a sperm progression velocity of 25–50 µm/s corresponds to 1.5–3.0 mm/min. Considering that the distance from the external cervical os to the oviductal ampulla is some 150 mm, theoretically a spermatozoon would take 50–100 min to swim the distance unaided. Adding a delay (minutes to days) in transport through the isthmus, it being a functional barrier and reservoir that is also influenced by the female hormonal status and ovulation, competent spermatozoa might arrive in the ampulla from perhaps 1 h up to several days after ejaculation at intercourse.
At insemination, sperm transport is further delayed because selection of spermatozoa from hypertonic liquefied semen into isotonic cervical mucus or isotonic preparation medium induces irreversible sperm tail-tip coiling and tail bending that reduces the proportion of motile spermatozoa as well as sperm velocity (see above) [Reference Holmes, Flanagan, Björndahl and Kvist43].
Considering artificial insemination, at intra-cervical insemination (ICI) the hypotonic shock occurs when spermatozoa in hypertonic liquefied raw semen are placed onto or into the isotonic cervical mucus. This will in turn delay sperm transport to the Fallopian tubes and affect their efforts to approach the oocyte. At intrauterine insemination (IUI) or Fallopian tube sperm perfusion (FSP), sperm distribution to the Fallopian tubes appears to be facilitated by the insemination technique. However, the hypotonic shock and its irreversible effect on sperm morphology and motility, would have occurred when the spermatozoa were selected from hypertonic liquefied semen into the isotonic preparation medium. Although flushed into the Fallopian tubes, their already impaired motility might hamper and delay the spermatozoa’s final approach to the oocyte [Reference Björndahl and Kvist14,Reference Holmes, Flanagan, Björndahl and Kvist43].
The human oocyte has a fertilization window of some 8–12 h after ovulation. It appears essential that a relatively small number of spermatozoa with full fertilizing potential are at hand in the Fallopian tubes to penetrate the cumulus and corona cells, the zona pellucida, and fuse with the oocyte to achieve fertilization within this short time window.
Normally around 70% of couples achieve pregnancy within six months after having randomly timed intercourse, so it can be argued that the conditions for sperm storage in these men and women are satisfactory. On the other hand, couples that do not achieve pregnancy spontaneously within a year of trying appear more likely to have impaired conditions for sperm storage: in theses couples the timing of ejaculation/insemination and ovulation seems to be of greater importance when treated with ART.
As illustrated in Figure 2.9, the proximal cauda of the epididymis and the isthmus of the Fallopian tube have evolved to be sites for sperm storage. In a man with full sperm production, the transit time through the epididymis is 2 days, with two main factors appearing essential to prolong sperm storage from 2 to 7–10 days: (a) low temperature (34°C), and (b) a normal testosterone-dependent environment in the proximal cauda epididymidis that causes low pH. Concordantly, the storage of spermatozoa in the Fallopian tube isthmus appears to be up to about 5 days if a normal estradiol-dependent environment creates a lower pH within the heavily convoluted lining of the isthmus where spermatozoa reside until ovulation [Reference Kölle, Flanagan, Björndahl and Kvist62]. The reduced pH is effectuated by carbonic anhydrase activity in the proximal cauda and in the isthmus.
Thus, the key players for sperm storage are (a) the gonadal production of testosterone and estradiol that, via blood perfusion, induce the low pH; and (b) the lower temperature achieved via a countercurrent blood flow system that achieves and maintains a temperature gradient from the 37°C core temperature of the body to 34°C at the base of the scrotum.
At ejaculation, the mammalian spermatozoon has not yet acquired full fertilizing capacity. The biochemical, molecular and physiological changes that the spermatozoon experiences within the female genital tract are collectively referred to as capacitation, a complex process that results in a spermatozoon fully competent for fertilization. During capacitation, changes occur in membrane properties, enzyme activities, and motility which together make spermatozoa responsive to stimuli that induce hyperactivated motility and, later, the acrosome reaction, and thereby prepare the spermatozoon for penetration of the egg investments prior to fertilization [Reference Mortimer11,Reference Mortimer61]. These changes are accompanied by the activation of cell signalling cascades in vivo or in defined culture media in vitro [Reference Salicioni, Platt and Wertheimer63–Reference Bosakova, Tockstein and Sebkova67], although the full nature of these signalling complexes, and their temporal and spatial activations, remain to be elucidated.
Hyperactivation is usually considered a part and expression of the capacitation process seen in all Eutherian mammals studied. However, the regulatory pathway that finally gives rise to an increase in flagellar Ca2+ and triggers hyperactivation can also operate independently from capacitation [Reference Suarez68,Reference Publicover, Harper and Barratt69].
Hyperactivation is characterized by high amplitude, asymmetrical flagellar bending, and robust assessment of hyperactivation needs parameters measured by CASA. Besides promoting the passage through the zona pellucida, hyperactivation may also facilitate release of sperm from the oviductal storage reservoir in the isthmus and may propel sperm through mucus in the oviductal lumen and the matrix of the cumulus oophorus [Reference Mortimer61].
The acrosome reaction is a ‘metamorphosis event’ that must be completed by the spermatozoa of many animal species prior to fusion with eggs [Reference Mortimer11]. In Eutherian mammals it follows capacitation and is triggered by the spermatozoon binding to zona pellucida. It involves multiple fusions between the sperm plasma membrane and the underlying outer acrosomal membrane, resulting in many small vesicles allowing release of the acrosomal content. In the mouse, this exocytosis is triggered by one of the three the zona pellucida glycoproteins, ZP3 (there is also a fourth ZP glycoprotein in the human ZP). Following binding of the spermatozoon to the zona ZP3 promotes a sustained influx of Ca2+ into the spermatozoon that is necessary for the acrosome reaction [Reference Florman, Jungnickel and Sutton70]. Among substances released during the acrosome reaction are hyaluronidase and acrosin. These are enzymes capable of degrading the cumulus mass and the zona pellucida, respectively. However, the hyaluronidase is apparently released at the zona pellucida, i.e. after the cumulus mass has already been traversed, and interestingly spermatozoa lacking acrosin can still fertilize, although they are less efficient [Reference Raterman and Springer71]. In a sea-snail, acrosin helps the spermatozoon open the oocyte-surrounding gel without acting as an enzyme. Thus, the presence of a molecule that can act as an enzyme does not necessarily mean that it actually has a physiologically important enzymatic effect.
The mammalian oocyte is surrounded by a thick glycoprotein layer, the zona pellucida, which the spermatozoon takes some 3–4 min to penetrate. Furthermore, around the zona pellucida is the huge cumulus mass, which is composed of about 50 layers of cumulus cells surrounded by the matrix produced by these cells. Hyaluronic acid is the predominant matrix macromolecule. The cumulus mass contains progesterone (secreted by the cumulus cells, which are follicular granulosa cells that underwent luteinization before ovulation) and nitric oxide, both of which induce calcium signalling within the spermatozoon [Reference Nagyova72]. Most spermatozoa fail to pass through, and become stuck in the cumulus, although exactly why some spermatozoa do not pass remains to be elucidated.
One factor could be lack of hyperactivated motility. Sperm penetration through the cumulus matrix, as well as through the zona pellucida (ZP), are dependent on hyperactivated motility, which is a type of motility characterized by high amplitude flagellar waves that generate powerful propulsive force, capable even of breaking covalent bonds [Reference Mortimer61]. After passage through the cumulus, the fertilizing spermatozoon must bind to the ZP (a largely species-specific process), undergo the acrosome reaction, and then penetrate through the matrix of the ZP. Thus, the ZP constitutes a barrier which, in most mammals, evolved to admit only spermatozoa of the same species. Following the acrosome reaction, new ligands are exposed on the equatorial segment of the sperm head by which the spermatozoon can bind to the oocyte plasma membrane after passing through the ZP [Reference Mortimer11].
In conclusion, the spermatozoon that finally fuses with the oocyte is one that underwent capacitation, developed hyperactivated motility, had the competence to pass the cumulus matrix, bore the right code to bind to the ZP, underwent the acrosome reaction, traversed the ZP, and finally showed the right code for binding to the oocyte membrane and was able to fuse with it.
Fertilization by fusion of the gametes is a fundamental mechanism and was the first evolutionary step towards sexual reproduction some 600 million years ago. After fusion, the sperm membrane and the oocyte membrane are contiguous [Reference Mortimer11]. The internal parts of the spermatozoon are then automatically inside and surrounded by the ooplasm. The contaminated outside of the sperm plasma membrane remains on the outside surface of the fertilized oocyte, although after ICSI it is also introduced into the ooplasm [Reference Kvist1,Reference Mortimer11,Reference Mortimer, Mortimer, Aitken, Mortimer and Kovacs73].
In biological terms ICSI, the injection of a spermatozoon directly into the ooplasm, is a new concept. From an evolutionary perspective, injection of spermatozoa (with their membranes intact) and some suspending medium means that the barriers of the oocyte are breached, raising the possibility of by-passing the natural barriers without physiological control, potentially exposing both the zygote and future generations to the entrance of compounds or organisms that otherwise would not have had access to the inside of the oocyte [Reference Mortimer, Mortimer, Aitken, Mortimer and Kovacs73]. This new evolutionary concept requires that we use controlled conditions, especially not using culture media that contain biological material from other species [Reference Kvist1,Reference Mortimer, Mortimer, Aitken, Mortimer and Kovacs73].
1) Factors for oocyte activation;
2) An intact haploid genome;
3) The centrosomes (needed for mitotic divisions in the new individual); and
4) Factors necessary for the initiation of placental development.
Besides DNA, the spermatozoon also brings proteins and mRNA to the oocyte, but the specific functions of these compounds still remain to be elucidated.
1. Activation of the Oocyte
The exact signals are unknown, but electrical events in the oocyte membrane and a rise in calcium in the ooplasm must occur. There is also the release of the cortical granules, which induce changes in the oocyte membrane so that it cannot fuse with other spermatozoa. Only if two spermatozoa are fusing with the oocyte almost simultaneously would polyspermic fertilization occur. Fertilization by two spermatozoa usually results in three pronuclei and a diandric ‘dispermic’ triploid zygote, which can develop to term (although human triploids always die within hours of birth). Fertilization by a diploid spermatozoon would create a ‘diplospermic’ diandric triploid zygote; this is very rare in vivo, more likely at IVF, and is avoided at ICSI by not injecting spermatozoa with large heads.
The oocyte then completes its second meiotic division, which is followed by the fusion of the male and female pronuclei and, some hours later, the first mitotic cell division. However, the oocyte can also be activated spontaneously, or by chemicals such as ethanol or even physical manipulation with a glass pipette. This capacity for such parthenogenetic development means that even if an oocyte divides into two cells there is no guarantee that it was fertilized by a spermatozoon.
2. An Intact Haploid Genome
Until recently, it was believed that the spermatozoon in most mammals contributed one centriole to the zygote and that this centriole duplicated into two that organized the first mitosis of the zygote. This was because it was believed that while the mammalian spermatid has two typical centrioles, the proximal and the distal, the distal was lost during late spermiogenesis.
Recently, however, it was demonstrated that the distal centriole seen in the elongating spermatid becomes remodeled in the mature spermatozoon into a functional-but-atypical centriole that is composed of microtubules surrounding earlier undescribed rods of centriole luminal proteins [Reference Fishman, Jo and Nguyen74]. Consequently, the ejaculated spermatozoon actually carries two functional centrioles, and both are transferred to the zygote. Both the proximal and the atypical distal centriole duplicate within the ooplasm and one of each origin forms a centrosome together with the oocyte pericentriolar material. The centrosomes organize ɣ-tubulin molecules in the ooplasm into long tubular threads starting at the centrosomes. The threads grow rapidly and can radiate like a ‘sperm aster’ throughout the ooplasm. The sperm asters anchor to the male and female pronuclei containing the paternal and maternal chromosomes. By contraction of the threads the sperm centrosome drags the pronuclei in close connection. At mitosis, one daughter centriole of the proximal and one of the atypical distal centriole forms the cell spindle of the first mitotic division. If this fails, the blastocyst will die.
The functional importance of the transition from a typical distal centriole to an atypical centriole is not yet understood [Reference Fishman, Jo and Nguyen74]. However, these new insights clearly provide new possibilities for diagnostics and therapeutic strategies for male infertility, and insights into early embryo development.
A sperm centriole that has been seriously damaged during sperm transfer might fail to sustain embryonic development. Fertilized oocytes that do not divide, or divide only slowly, could have a damaged sperm centriole. Even dividing fertilized oocytes chosen for embryo transfer could be destined to die due to sperm centriolar failure. The anti-cough drug noskapin, and also diazepams, interfere with sperm aster formation, but the extent to which drugs that interfere with centrosome function might have affected human fertility is not known.
The centrosome is a self-replicating organelle which in most mammals (including humans) is inherited from the male. Interestingly, in mice and other rodents, the zygote does not receive a centriole from the spermatozoon, but the whole centrosome (two perpendicularly oriented cylindrical centrioles and the pericentriolar material, or PCM) comes from the oocyte, a fundamental difference in mammalian evolution [Reference Schatten75]. Hence, the centriole is a specialized expression of the centrosome that serves as the main microtubular organizing centre in the zygote and a regulator of cell-cycle progression. It can initiate the aggregation, orientation and function of certain intracytoplasmic threads or tubules for movement and transport, and can also reproduce and is claimed to carry its own RNA-genome, necessary for aster formation for example. A centriole can in turn give rise to a cilium or a flagellum as the sperm tail. In some cells, the centrosome forms many centrioles (basal bodies), each giving rise to a cilium, as in ciliated cells in, e.g., the Fallopian tubes and in the respiratory epithelium. The round spermatid carries one typical centrosome, which has given rise to two centrioles, the proximal and distal centrioles in the connecting piece of the elongating spermatids. The distal centriole gives rise to the sperm flagellum, the basic element of the tail, and can be seen posterior to the connecting piece by transmission electron microscopy in the elongating spermatids. The proximal centriole may have helped in the organization of the chromosomes in the nucleus and shaping of the head by the manchette [Reference Lehti and Sironen76].
4. Factors Necessary for Initiation of Placental Development
In placental mammals like humans, the nutritional capacity of the oocyte lasts for a few days only. Chorionic villi grow out from the surface of the embryo in all directions, and upon meeting the endometrium these villi actively transform the endometrium into the maternal part of the placenta-to-be. Thus, the embryo plays the active role in placentation. The acceptance of a foreign individual intermingling with the tissue of the host (here the endometrium) was a step taken in evolution and its full consequences are not fully elucidated, e.g. for acceptance of invasive cancer growth.
Two sperm nuclei in a frog oocyte will result in a frog. Two oocyte nuclei in a frog oocyte also will result in a frog. In a placental mammal the situation is different: two sperm nuclei in a mouse oocyte will result in mainly placental tissue. The human equivalent has been known for several years, although its implications were not understood. Two sperm nuclei in a human oocyte result in a hydatidiform mole. In contrast, two oocyte nuclei in an oocyte result in mainly embryonic tissue without initiation of proper placental tissue and the embryo will die. The mechanism by which the spermatozoon initiates placental growth is not understood. Genomic imprinting, i.e. different inactivation of genes by epigenetic control within the male or female gonad, is a favourable candidate for this mechanism.
Evolution of placental animals thus has given the spermatozoon a ‘new’ important function. Paternal factors thus secure a passage of nutrients and other vital substances to the fetus while the block for placental formation within the oocyte genome protects the female from offspring achieved through parthenogenesis. A consequence of this is that research on factors contributing to subfertility must also consider the possibility that damages to the sperm and its messages might disturb placental formation and function and therefore also be involved in problems related to growth and development a long time after fertilization.
Because sperm mitochondria were already ubiquitin-tagged in the testis, marking them and their DNA for destruction after entry into the oocyte [Reference Sutovsky and Song23], in evolutionary terms mitochondria are inherited through the female germ cell line. While there are reports suggesting that mutated sperm mitochondrial DNA may escape destruction and cause mitochondrial disease in the offspring, it seems to be an extremely rare event.
This chapter has summarized how nature creates, stores and transfers spermatozoa, and how those spermatozoa with intact DNA, mitochondria, centrioles and placental factors give rise to healthy progeny. There are challenges for ART labs to comprehend and always act in concordance with nature, especially the early critical moment of collecting and selecting spermatozoa for use in ART treatments. Nature immediately selects motile spermatozoa from the isotonic, zinc-rich prostatic fluid into the isotonic cervical mucus. But before the human spermatozoon reaches the oocyte in the ART lab it experiences two major non-physiological challenges that can affect its full fertilizing potential: (a) exposure to seminal vesicular fluid (causing chromatin zinc-deficiency and a vulnerable chromatin with increased risk for oxidative DNA damage); and (b) exposure to an osmotic roller-coaster that causes sperm ATP-depletion, tail coiling, impaired sperm motility and low sperm density (as measured in g/ml, not to be confused with sperm concentration), that affect sperm functional properties and sperm selection procedures by swim-up and density gradient centrifugation. These two unphysiological challenges are unintentionally forced upon the spermatozoa when collected as a whole ejaculate and then subjected to diagnostic assessments or sperm selection for ART [Reference Houska, Flanagan, Björndahl and Kvist38–Reference Holmes, Flanagan, Björndahl and Kvist43].
Obviously, the causes and consequences of these stresses need to be considered and eliminated in order to improve our skills in diagnostics and assisted reproduction. So, for the future, we should consider paying more attention to replicating biophysical and biochemical conditions, as well as practical handling steps, that better support sperm physiology.