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ANATOMY AND PHYSIOLOGY OF MALE REPRODUCTION

HORMONAL CONSIDERATIONS TESTICULAR ANATOMY SEMINIFEROUS TUBULES THE EPIDIDYMIS

Seminiferous Tubules Straight Tubules Rete Testis Efferent Ductules Epididymis Vas Deferens Urethra

Compared to other species, human males have relatively poor sperm producing capacity and human testicular function is very sensitive to a wide variety of environmental insults. This may be related to the human (upright) posture and hydrostatic pressure on venous testicular outflow, or other unknown factors, but it is necessary for clinicians to be aware of the high incidence of sub fertility in men. Perhaps it is a reflection of the incredible ability of humans to adapt the environment to promote their own survival or the expectation that fertility should be nearly spontaneous, but many human couples seek evaluation for infertility. The human male reproductive system includes the hypothalamic-pituitarytestis axis as well as the i. Epididymis, ii. Vas deferens, iii. Seminal vesicles iv. Prostate v. Urethra. Production of spermatozoa requires approximately 3 months from the initial mitotic divisions through the myriad changes readying sperm for ejaculation and fertilization. Highlights of this transformation include 1. The unique environment created within the testis for spermatogenesis to occur; 2. Preservation of a set of stem cells relatively resistant to external injury and able to produce rapidly proliferating germ cells destined to become spermatozoa; 3. Meiosis, that results in formation of the haploid gamete;

4. The dramatic differentiation of the prospective gamete in a form that is specialized to transport chromosomal material in a structure ideally suited for transit of the female reproductive tract. The spermatozoon resulting from this complex process assumes its final shape and size in the testis. In the normal state, it also acquires the ability to fertilize as well as a capacity for motility in the epididymis. Unfortunately, the mechanisms by which the epididymis exerts these changes on the traversing spermatozoon and the actions of the human reproductive tract after relief of chronic obstruction remain largely unknown. 1. HORMONAL CONSIDERATIONS An appropriate hormonal milieu must exist for the reproductive organs to produce, mature and transport the highly specialized male gamete to the ejaculatory duct. The entire system of hormone balance is initiated by the pulsatile hypothalamic release of GnRH (gonadotropin-releasing hormone). Pituitary LH (luteinizing hormone) secretion is determined by GnRH pulses from the hypothalamus that occur approximately every two hours and are carried via a venous portal network to the pituitary. This hypothalamohypophyseal portal connection allows an exact synchrony of GnRH and LH pulse secretion. FSH (follicle-stimulating hormone) secretion is also stimulated by GnRH, but FSH and LH are differentially regulated by hormonal and other factors that are poorly understood. The factors influencing FSH secretion are produced by Sertoli cells and other components of the testis that probably includes peptides of the inhibin and activin families. Within the testis, LH stimulates Leydig cell synthesis of testosterone. Testosterone production by the Leydig cell provides locally high intratesticular concentrations of this hormone that stimulates spermatogenesis. Testosterone concentrations in peripheral blood of men change dramatically during the life cycle. Testosterone reaches a maximum concentration during the second or third decade of life, then reaches a plateau, and declines thereafter. Additionally, annual and daily rhythms in testosterone concentration occur, typically with a testosterone peak in the early morning. Other, irregular fluctuations in testosterone concentration may also be detectable in peripheral blood. Testosterone is normally aromatized in peripheral tissue to estrogens. Excessive testosterone levels, associated with gonadotropin, clomiphene citrate or flutamide treatment, may paradoxically result in increased feminization from conversion of androgens to estrogens by aromatase. Similarly,

increased aromatase activity is associated with alcoholism and chronic liver disease, as well as testis tumors. Accurate clinical assessment of the pituitary gonadotropins LH and FSH must take into account their pulsatile release. During clinical research studies, three serum samples are obtained, one every 30 minutes, and the sera pooled for accurate determination of mean gonadotropin levels. This process is usually not necessary in clinical practice, but the clinician should be aware of the potential for LH and FSH peaks to be measured in a single gonadotropin determination, and perform repeat evaluation if LH and FSH hormone levels are both elevated. Testosterone levels may be decreased in the late afternoon or evening. Interpretation of serum testosterone levels should take the diurnal secretion of this hormone into account. Prolactin, another pituitary hormone, may affect fertility by decreasing LH production, resulting in a decrease in testosterone and subsequently, decreased libido. The release of prolactin is mediated by dopamine, and the dopamine antagonist bromocriptine will ameliorate the antifertility effects of hyperprolactinemia. Testosterone is converted intracellularly within most androgen-sensitive organs to dihydrotestosterone. Function of the prostate, seminal vesicles, vas deferens, and other sex accessory organs are all androgen-dependent. The degree to which partial androgen deprivation in the hypogonadal man affects the function of these organs is unknown. Furthermore, the effects of "low-normal" serum testosterone levels on these organs and a man's fertility potential are unknown. Abnormally elevated serum testosterone levels are peripherally converted by the aromatase enzyme to estrogens. In addition, chromosomal abnormalities such as Klinefelter's syndrome (xxy), and some testicular tumors have elevated aromatase levels. Some obese patients may also have increased aromatase activity, since aromatase levels are high in adipose tissue, as well as fat. Therefore, hormonal evaluation of the infertile man should include determination of serum LH, FSH, testosterone and prolactin. For men with clinical gynecomastia, serum estradiol should be measured. 2. TESTICULAR ANATOMY The human testis is an ovoid mass that lies within the scrotum. The average testicular volume is 20 cc in healthy young men and decreases in elderly men. In Asian men, testes tend to be smaller. Normal longitudinal length of the testis is approximately 4.5 to 5.1 cm.

The testicular parenchyma is surrounded by a capsule containing blood vessels, smooth muscle fibers and nerve fibers sensitive to pressure. The functional role of the testicular capsule is unknown, but may relate to movement of fluid out through the rete testis or control of blood flow to the testis. The testis contains seminiferous tubules and interstitial cells. The tubules are segregated into regions by connective tissue septa. The seminiferous tubules are long V-shaped tubules, both ends of which usually terminate in the rete testis. Measurement of testicular size is critical in the evaluation of the infertile man, since seminiferous tubules (the spermatogenetic region of the testis) occupy approximately 80% of testicular volume. So, a rough estimate of spermatogenic cell capacity is provided by assessment of testicular size. Testicular consistency is also of value in determining fertility capacity. A soft testis is likely to reflect degenerating or shrunken spermatogenic components within the seminiferous tubules. The seminiferous tubules drain toward the central superior and posterior regions of the testis. The rete testis, that has a flat cuboidal epithelium. The rete coalesces in the superior portion of the testis, Just anterior to the testicular vessels, to form 5-10 efferent ductules. These efferent ducts leave the testis and travel a short distance to enter the head, or caput region of the epididymis. The efferent ducts coalesce in a somewhat variable pattern within the caput epididymis to form a single epididymal tubule. The artery to the testis is specialized in that it is highly coiled and intimately associated with a network of anastomotic veins that form the pampiniform plexus. The counterflowing vessels are separated only by the thickness of their vascular wall in some areas. This vascular arrangement facilitates the exchange of heat and small molecules, including testosterone. The transport of testosterone is a concentration-limited, passive diffusion process in men. The counter-current exchange of heat in the spermatic cord provides blood to the testis that is 2 to 4 C lower than rectal temperature in the normal individual. A loss of the temperature differential is associated with testicular dysfunction in humans with idiopathic infertility, as well as men with varicocele or cryptorchidism. Whether elevated testicular temperature causes or is simply a reflection of testicular dysfunction is unknown. Only the association between elevated testicular temperature and seminiferous failure has been demonstrated. In the distal inguinal canal, 50% of men will have a single testicular artery identifiable under l0 x power magnification dissection of the cord, with 30% of men having two arteries and 20% with three arteries.

The venous system is somewhat unique because the spermatic veins are thinwalled, poorly muscularized, and lack effective valves except at the inflow points into the inferior vena cava or the renal vein. The right spermatic vein usually drains into the vena cava. The left spermatic vein drains into the left renal vein. The renal vein on the left side is thought to have a higher intraluminal pressure because the vein is compressed as it passes between the superior mesenteric artery and the aorta. This "nutcracker effect" may impair flow through the left renal and spermatic veins, especially in young men with limited retroperitoneal fat. The differential anatomy of the left and right spermatic veins is thought to explain, at least in part, the higher prevalence of varicoceles on the left side. The exact mechanism by which varicoceles cause infertility is unknown. In animal models, varicoceles are associated with increased blood flow to the testis and increased interstitial fluid in the testis. These two findings may impair regulation of testicular temperature and decrease intratesticular concentrations of testosterone or other local factors important for spermatogenesis. 3. SEMINIFEROUS TUBULES The seminiferous tubules provide a unique environment for the production of germ cells. The structures involved in this process include germinal elements and supporting cells. The supporting cells include the peritubular cells of the basement membrane and the Sertoli cells. The germinal elements comprise a population of epithelial cells, including a slowly dividing primitive stem cell population, the rapidly proliferating spermatogonia, spermatocytes undergoing meiosis, and the metamorphosing spermatids. The seminiferous tubule also produces an environment known as "the blood-testis barrier". The testis is unique in that the differentiating germ cells are potentially antigenic, and recognizable as foreign; however, little immunological reaction is usually detectable within the testis. Developmentally, the testis develops from the undifferentiated gonad. These primitive germ cells are referred to as gonocytes after the gonad differentiates into a testis by forming seminiferous cords. At this time, the gonocytes are located in a central position within the seminiferous cords. They are subsequently classified as spermatogonia after the gonocytes have migrated to the periphery of the tubule. From birth to approximately 7 years of life, there appears to be very little morphological change within the human testis.

From 7 to 9 years of life, mitotic activity of gonocytes is detectable, with spermatogonia populating the base of the seminiferous tubule in numbers equal to those of the Sertoli cells. There appears to be little further morphological change in spermatogonia until spermatogenesis begins at the time of puberty. Further information regarding the maturation of gonocytes and their migration to the base of the seminiferous tubule, including the factors that may be responsible for these changes, may provide greater insight into the effects of cryptorchidism on fertility and impact on the appropriate timing of intervention for treatment of cryptorchidism. 4. THE EPIDIDYMIS Spermatozoa in the unobstructed testis are not motile and are incapable of fertilizing ova. Spermatozoa become functional gametes only after they migrate through the epididymis and undergo an additional maturation process, thereby acquiring the capacities for both progressive motility and fertility. The function of the obstructed epididymis and its effects on maturation of spermatozoa may be very different from what is observed in the unobstructed state. Anatomically, the epididymis can be divided into three regions: 1. The caput, 2. The corpus 3. The cauda epididymis. However, these anatomical divisions have been defined based on findings in animals, not in humans. The human epididymal epithelium is relatively homogeneous as viewed under the microscope, and grossly, the epididymis does not have the same distinct gross anatomical subdivisions that are easily seen in the rat, rabbit and other animals. Unfortunately, there is little information available regarding the functional diversity of these three regions of the human epididymis. The data that exist on human epididymal function are almost entirely derived from observations of men after relief of chronic epididymal obstruction. In humans, the epididymis receives its blood supply from two sources: Two branches of the testicular as well as the deferential vessels that travel along the vas deferens. Clinically, the blood supply becomes important after a vasectomy with interruption of the vasal vessels.

At the time of vasectomy reversal, the testicular side of the vas deferens is then supplied by branches off the testicular vessels, which travel either through collaterals, or along the entire length of the epididymis. Similarly, if division of the vasal vessels occurs accidentally during vasography, and a secondary obstruction to the vas deferens (and vasal vessels) is found and addressed in the groin, then the intervening segment of vas deferens can be totally devascularized. The viability of the vas should be considered prior to vaso-vasostomy in this situation.Observations by Silber regarding the fertility of men who have undergone bilateral vasal anastomosis to the vasa efferentia, indicate that in the obstructed human male reproductive tract, some sperm may acquire motility and fertilizing ability without passing through the epididymis. Although not absolutely required for male fertility, the functional importance of the human epididymis is strongly supported by several other observations regarding men who have undergone intervention for the relief of chronic epididymal obstruction. In 1990, Silber reported fertility results for men who underwent vasoepididymostomy at the level of either the caput or corpus epididymis. For both groups of men, the patency rate was approximately 70%. For men who underwent anastomosis at the corpus level, 72% of men achieved pregnancy with their partners, whereas only 43% of men were able to impregnate their wives after vasoepididymostomy at the caput level.Therefore, the presence of a longer length of epididymis appears to promote fertility after relief of chronic obstruction. Our studies in men with congenital absence of the vas deferens or other surgically unreconstructable obstruction of the vas have indicated that the longer the segment of epididymis present, the greater the likelihood of pregnancy with sperm obtained from these men. Almost all of the information regarding human epididymal function are derived from observations of men who have undergone surgical reversal of long term reproductive tract obstruction. The results of several published observations of men with unobstructed reproductive tracts were indicated the fertility potential of sperm from the caput region of the obstructed human epididymis is minimal.The exact fate of unejaculated epididymal spermatozoa in humans is also unknown. Some sperm have been documented to be phagocytosed by macrophages in the epididymis, and sperm have been detected in urine. However, the mechanism by which quantitative clearance of sperm occurs from men during periods of sexual abstinence is unknown.

In normal, unobstructed systems, the majority of spermatozoa taken from the vasa efferentia and diluted in a physiologic solution is immotile or exhibit only weak tail movements. These observations in the unobstructed human epididymis are very different from what is seen after relief of chronic epididymal obstruction. Our observations confirm those of several investigators, that, although the motility of ejaculated spermatozoa is initially poor following vaso-epididymostomy at the level of the caput epididymis, sperm motility may improve greatly up to 1 1/2 years after vasoepididymostomy. These findings suggest that following vasoepididymostomy the caput epididymis or vas deferens may undergo compensatory adaption over time and support sperm motility maturation despite exposure of sperm to a shortened length of epididymis. In animal models, vasal obstruction causes changes in the intrinsic motility of fluid through the epididymal lumen. These changes are not completely reversed by technically successful vasovasostomy. It is not known what effect the apparent change in epididymal contractility may have on epididymal function. Studies on laboratory animals describe many changes that occur to sperm during epididymal transit. These include a change in net sperm surface charge, addition and alteration of membrane proteins, alterations in sperm lectin-binding properties, changes in immunoreactivity and iodination characteristics, acquisition of an increased capacity for glycolysis, modification of adenylate cyclase activity, alterations in cellular phospholipid and phospholipid-like fatty acid content and an increased ability to adhere to the zona pellucida of the egg. Whether similar modifications occur in human spermatozoa during epididymal migration is unknown. Biochemical changes observed in human spermatozoa during epididymal transit involve the formation of disulfide bonds within the sperm nucleus and tail and the oxidation of sperm membrane sulfhydryl groups. These changes are thought to provide improved structural integrity to the sperm membrane. The changes in structural integrity of sperm may be necessary for the development of progressive motility and successful penetration of eggs. Protein secretion and the storage capacity of the epididymis has been shown to be profoundly affected by changes in epididymal temperature in animals. Some have even postulated that the driving force for evolution of the scrotal location of testes is to have the epididymis maintained at a temperature below that of body core temperature, in the scrotum. Whether the functions of the human epididymis are similarly affected by body temperature is unknown.

The potential influence of temperature on epididymal function in man may be an important consideration in explaining the relationship between varicocele and male infertility. If temperature significantly affects human epididymal function, then it could explain improvements in semen parameters that may occur less than three months (one full cycle of spermatogenesis) after varicoceletomy. The human spermatozoon is approximately 60 m in length. Normal forms have an oval sperm head, 4.5 m long and 3 m wide, consisting principally of a nucleus, which contains the highly compacted chromatin material, and the acrosome, a membrane-bound organelle that covers 40-70% of the surface of the sperm head and contains the enzymes required for penetration of the outer vestments of the egg prior to fertilization. The middle piece of the spermatozoon is a highly organized segment consisting of helically arranged mitochondria surrounding a set of outer dense fibers and the characteristic 9 + 2 microtubular structure of the sperm axoneme. The mitochondria contain the enzymes required for oxidative metabolism and the production of adenosine triphosphate (ATP), the primary energy source for the cell. Absence of some components of the microtubules is associated with immotile cilia in the sinus and pulmonary tracts, with resultant pulmonary and sinus infections. The association of sperm immotility with upper respiratory infections (Young's syndrome) due to ciliary dysfunction or absent dynein crossarms between the microtubules (Kartagener's syndrome) resulting in bronchiectasis and nonmotile sperm have been well documented. Other syndromes of ciliary dysfunction have recently been described in children with recurrent sinusitis (ciliary dyskinesia). It is yet to be demonstrated whether the boys affected with the childhood manifestations of ciliary dyskinesia will suffer from infertility problems after puberty. The highly specialized structure and physiology of the spermatozoon is marvelously suited for its single purpose: to carry genetic material to the egg during reproduction. The rate of transport of fluid through the vas deferens is not known in the human. Just prior to ejaculation, the testes are brought up close to the abdomen and fluid is rapidly transported through the vas deferens toward the region of the ejaculatory ducts and subsequently into the prostatic urethra. After ejaculation, intravasal fluid is transported back toward the epididymis and occasionally into the seminal vesicles as well. The retrograde transport of sperm to the seminal vesicles has been documented by videoradiography during ejaculation after vasography. The return of sperm to the seminal vesicles after ejaculation may help explain the prolonged presence of sperm in the ejaculate for some men after vasectomy.

The ejaculatory ducts enter the prostatic urethra just lateral to the verumontanum. In the case of obstruction of the ejaculatory ducts, resection of the floor of the prostate should be performed just lateral to the midline and superior to the verumontanum. Vasography can be performed prior to transurethral resection (TUR) of the ejaculatory ducts with placement of methylene blue in the vasography fluid. This allows the surgeon to cystoscopically identify the lack of flow of dye and confirm relief of obstruction of the ejaculatory ducts during TUR. The ejaculate consists of components from different accessory organs. Each of these organs and the characteristics of the fluid that they produce is listed below. These characteristics can be used clinically to evaluate ejaculatory dysfunction.

Spermatogenesis

Fig (1) A reduced number of spermatozoa, predominating malformed spermatozoa or reduced and inefficient motility may be the cause for disturbed fertility or infertility of a patient.

(A) Sector of the germinal epithelium in the seminiferous tubule. Drawing on the basis of a semithin section. 900 (B) Sertoli cells divide the germinal epithelium in a basal and adluminal compartment. Arrows indicate the transport of substances only to the basal compartment, via the Sertoli cell into the adluminal compartment, via the Sertoli cell into the lumen. Fig(2)

(A) The storage of lipid droplets of different size and composition in the Sertoli cells correlates to the age of the man. The Sertoli cell represent a "biological clock" of the testis. (B) Seminiferous tubule with marked clones of germ cells. Drawing on the basis of a semithin section. 300 Fig(3)

(A) Section of the germinal epithelium with multilayered spermatogonia. 52 years old infertile patient with arrest of spermatogenesis at the stage of spermatogonia. Drawing on the basis of a semithin section. 600 (B) Arrest of spermatogenesis at the stage of A pale type-spermatogonia. 37 years old infertile patient with arrest of spermatogenesis at the stage of spermatogonia. Drawing on the basis of a semithin section. 600 (C) Tumour cells in the basal compartment of the germinal epithelium dislocate a pale type-spermatogonia. 33 years old patient. Drawing on the basis of a semithin section. 600 (D) Megalospermatocytes do not complete meiosis. 37 years old patient with impaired fertility. Drawing on the basis of a semithin section. 600 (E) Arrest of spermatogenesis at the stage of primary spermatocytes. 34 years old patient with impaired fertility. Drawing on the basis of a semithin section. 600 (F) Arrest of spermatogenesis at the stage of immature spermatids. 52 years old patient with impaired fertility. Drawing on the basis of a semithin section. 600. Fig(4)

(A) Steps of spermatid differentiation: (1) Immature spermatid with round shaped nucleus. The acrosome vesicle is attached to the nucleus, the tail anlage fails contact to the nucleus. (2) The acrosome vesicle is increazed and flattened over the nucleus. The tail contacted the nucleus. (38) Acrosome formation, nuclear condensation and development of tail structures take place. The mature spermatid (8) is delivered from the germinal epithelium. Semi-schematic drawing on the basis of electron micrographs. From Ref. [14]. (B) Development of a giant spermatid by confluence of double headed spermatids of a clone. The giant spermatid remains in contact with the Sertoli cell. Drawing on the basis of electron micrographs. From Ref. [9]. (C) Differentiation of acrosomeless spermatozoa. The nuclear condensation and the development of tail structures is not disturbed. The acrosome, however, fails to establish contact to the nucleus of the spermatid and remains in the Sertoli cell cytoplasm. Drawing on the basis of electron micrographs. From Ref. [9].

Fig(5)

(A) Development of headless spermatozoa. Only the proximal centriole contacts the basal plate of the nucleus of the spermatid. The distal centriole is separated and develops the headless flagellum. Drawing on the basis of electron micrographs. From Ref. [9]. (B) Loss of the mitochondrial sheath during spermiogenesis. The spermatozoon is immotile. Drawing on the basis of electron micrographs. From Ref. [9]. (C) Development of malformed fagellar structures. Drawing on the basis of electron micrographs. From Ref. [9]. (D) Multiple malformations of spermatids. Drawings on the basis of electron micrographs from testicular specimen of several patients aged 4385 years. The testes were removed by surgery as an additive treatment of prostatic cancer.

Fig(6)

(A) Microvasculature of the intertubular space. Co-cells encase the capillaries, Leydig cells and the seminiferous tubule. Modified from Ref. [20]. (B) The human spermatozoon. (1) Light microscopical aspect, (2) Virtual preparation of the spermatozoon showing the acrosome, the nucleus and nuclear envelopes, the mitochondrial sheath of the main piece of the flagellum, (3 11) Cross sections of the human spermatozoon of different levels indicated in (12) longitudinal section of the human spermatozoon. Semi-schematic drawing on the basis of electron micrographs. From Ref. [14].

Fig(7)

Introduction Throughout spermatogenesis, Multiplication, Maturation Differentiation of germ cells Results in the formation of the male gamete. The understanding of spermatogenesis needs detailed informations about the, 1. Organization of the germinal epithelium, 2. The structure and function of different types of germ cells, 3. Endocrine functions, 4. Paracrine cells mechanisms, 5. Intratesticular environment, 6. Extratesticular regulation of spermatogenesis. Normal germ cells must be discriminated from malformed, apoptotic and degenerating germ cells and tumor cells. Identification of the border line between normal and disturbed spermatogenesis substantiate the diagnosis of impaired male fertility. The profound knowledge of the complicate process of spermatogenesis and all cells or cell systems involved with is the prerequisite to develop concepts for therapy of male infertility or to handle germ cells in the management of assisted reproduction. Starting from a self-renewing stem cell pool, male germ cells develop in the seminiferous tubules of the testes throughout life from puberty to old age. The complete process of germ cell development is called spermatogenesis. Subdivisions are Spermatogoniogenesis, Meiosis, Spermiogenesis, Spermiation. The products of spermatogenesis are the mature male gametes, namely the spermatozoa. The light microscopical evaluation of the ejaculate permits (1) Evaluation of the number of spermatozoa, Shape Motility patterns Assessment of other cellular components. All these provide the first information about the success of spermatogenesis. Yet, the standard evaluation of the ejaculate does not provide in many cases sufficient information about the defects of spermatogenesis. A more thorough evaluation of the ejaculate may disclose a variety of disturbances originating in the different steps of spermatogenesis and may shed

light on disturbed testicular functions or even disclose in the presence of early testis cancer. Biopsies of the testes may be necessary to obtain valid informations about the quality of spermatogenesis or for exclusion of early testis cancer. Spermatogenesis depends from intratesticular and extratesticular hormonal regulatory processes and functions of the intertubular microvasculature, the Leydig cells and other cellular components of the intertubular space. The complex situation may be elucidated step by step. A reduced number of spermatozoa, predominating malformed spermatozoa or reduced and inefficient motility may be the cause for disturbed fertility or infertility of a patient. (2) Organization of the testis The human testes are two organs of the shape of rotation ellipsoids with diameters of 2.5 4 cm engulfed by a capsule (tunica albuginea) of strong connective tissue. Thin septula testis (Fig. 1A) divide the parenchyma of the testis in about 370 conical lobules. The lobules consist of the seminiferous tubules and intertubular tissue, containing groups of endocrine Leydig cells and additional cellular elements. The seminiferous tubules The seminiferous tubules are coiled loops (Fig. 1B). Their both ends open into the spaces of the rete testis. Structure of the seminiferous tubules The seminiferous tubule consists of the germinal epithelium and the peritubular tissue (lamina propria)(Fig. 1C). The mean diameter of a seminiferous tubule is about 180 m, the height of the germinal epithelium measures 80 m and the thickness of the peritubular tissue is about 8 m. (3)Rete testis The fluid secreted by the seminiferous tubules is collected in the rete testis and delivered to the excurrent ductal system of the epididymis. (4) The germinal epithelium (Fig. 2A) Consists of cells that include different developmental stages of germ cells, namely spermatogonia, primary and secondary spermatocytes and spermatids. These are located within invaginations of Sertoli cells. The prismatic Sertoli cells are connected by specialized zones of tight junctions of cellular membranes separating the germinal epithelium in a basal and an adluminal compartment (Fig. 2B). These specialised zones, the so-called "tight junctions" form the blood-testis barrier of the testis.

During maturation the germ cells pass this barrier entering the adluminal compartment where they find protection from diffusion of extraneous substances. Sertoli cells, investigated in histological sections, exhibit increasing amounts of lipid droplets in correlation to advanced age being an indicator of the "biological clock" of the testis (Fig. 3A). (5) Further functions are attributed to Sertoli cells 1. Sustentacular and nutritive functions for the germ cells. 2. Organization of the delivery of mature spermatids into the tubular lumen (spermiation). 3. Production of endocrine and paracrine substances for the regulation of spermatogenesis. 4. Secretion of androgen binding protein (ABP) for the maintenance of epithelia of the excurrent duct system. 5. Interaction with the intertubular endocrine Leydig cells. The peritubular tissue (lamina propria of seminiferous tubules) consists of about five layers of myofibroblasts with intermingled connective tissue ground substance. (6) Transport of the immotile spermatozoa to the rete testis The myofibroblasts cause peristaltic contractions of the seminiferous tubule giving rise to transport of the immotile spermatozoa to the rete testis. The thickness of the peritubular tissue normally is about 8 m. In cases of disturbed spermatogenesis the peritubular tissue may be thickened by connective tissue ground substance up to 12 m and more. (7)Spermatogenisis Spermatogenesis is the process by which a complex, interdependent population of germ cells produces spermatozoa. Spermatogenesis begins at puberty after a long preparatory period of "prespermatogenesis" in the fetus and the infant. Three major stages can be distinguished: 1. spermatogoniogenesis, 2. maturation of spermatocytes 3. spermiogenesis, Which is the cytodifferentiation of spermatids? Spermatogoniogenesis Several types of spermatogonia are distinguished by their position in the basal part of the germinal epithelium, their morphology and stainability of nuclei: A pale type-, A dark type- A type spermatogonia belong to the stem cell pool of spermatogenesis B type-spermatogonia B type-spermatogonia represent the onset of germ cell development up to spermatids. Spermatogonia multiplicate continuously in successive mitoses. Spermatogonial cell divisions are usually incomplete.

The daughter cells remain interconnected by cytoplasmic bridges so that a clone derived from one stem cell forms a syncytium of cells. Syncytial connections are maintained through spermatogonial and spermatocytic stages and are dissolved only in advanced phases of spermatid development. It is thought that the formation of these clones are the basis for the synchronuous development of germ cells (Fig. 3B). A type spermatogonia Both A type spermatogonia are necessary for intact spermatogenesis. In reduced spermatogenesis A dark-type spermatogonia are often absent. Of course, in the absence of both types of spermatogonia, no spermatogenesis takes place and the germinal epithelium consists of Sertoli cells only. congenital Sertoli cell-only Syndrome Spermatogonia may be absent from birth. acquired Sertoli cell-only Syndrome Destroyed by different noxes, e.g. x-radiation. (8) B-type spermatogonia In cases of disturbed ability of spermatogonia to develop B-type spermatogonia the number of A pale type spermatogonia increases and bi- or multilayered groups (Fig. 4A) of spermatogonia in the basal compartment are formed without further developed germ cell stages. (9) This aspect represents an arrest of spermatogenesis at the stage of spermatogonia (Fig. 4B)[9]. (10) The barrier of Sertoli cells can not normally be passed by A typespermatogonia. Under special conditions, e.g. intratubular tumor cells, the barrier is interrupted and spermatogonia are dislocated into the adluminal compartment where they disintegrate. (11) In the basal compartment of the seminiferous tubules tumour cells may be found. In semithin sections they differ noticeable from spermatogonia because of the their larger size, the prominent nucleolus, increase glycogen content and a clear peripheral border (Fig. 4C). (12) In paraffin sections the detection of single tumour cells may be difficult and the PLAP (Placental alkaline phosphatase)-reaction is required to demonstrate a characteristic dark border. Occasionally hypospermatogenesis is caused by the presence of neoplastic cells in the basal compartment of the germinal epithelium along the basal lamina. These basally situated neoplastic cells in the seminiferous tubules are characteristic of carcinoma-in-situ. They appear to be the stem cell population for most germ cell tumours including both seminomatous and teratomatous tumour types. Sporadic tumour cells may be found within tubules in association with active spermatogenesis, but as the neoplastic cells increase in number, spermatogenesis ceases and the remaining spermatogonia become detached and are released into the tubular lumen.

After further proliferation of the neoplastic cells, these also appear in the lumen of the tubule or penetrate the peritubular tissue giving rise to the development of intertubular tumour cell clusters. Meiosis of spermatocytes The stage of meiosis is manifested through changes in chromatin configuration in the nucleus after the last spermatogonial division. Cells in meiosis are called spermatocytes. As the process of meiosis comprises two divisions, 1. primary spermatocytes cells before the first division 2. secondary spermatocytes before the second division The primary spermatocytes are the largest germ cells of the germinal epithelium (Fig. 1C). The aspect of their nuclear chromatin represents the meiotic stages. Meiosis of spermatocytes starts with the leptotene stage of prophase already in the basal compartment of the germinal epithelium. After passing the Sertoli cell barrier, spermatocytes reach the adluminal compartment and continue with the further prophase stages, namely Zygotene stage Pachytene stage, Diplotene stage. During the prophase the reduplication of DNA, the condensation of chromosomes, the pairing of homologuous chromosomes and the "crossing over" take place. After division the germ cells become secondary spermatocytes. They undergo no DNA-replication and divide quickly to the spermatids. The two maturation divisions of each spermatocyte result in four haploid cells, namely the spermatids. These differentiate into mature spermatids, a process called spermiogenesis which ends when the cells are released from the germinal epithelium. At this point, the free cells are called spermatozoa. Many defects of meiosis are known indicating the vulnerability of this complicate process. Apoptotic spermatocytes are frequent. (13) Megalospermatocytes In some cases very large spermatocytes, so called megalospermatocytes (Fig. 4D) appear. In these cells asynapsis of homologuous chromosomes occurs and the cells become abortive. Genetic disorders may cause these defects. Often, arrest of spermatogenesis at the stage of primary spermatocytes appears without any special aspect of changed morphology of the cells. The primary spermatocytes border the lumen of the seminiferous tubule and do not develop further (Fig. 4E). They disintegrate and spermatids are missing. Spermiogenesis

During the cytodifferentiation of spermatids the following three processes take place: (fig 5A) Condensation of the nuclear chromatin to about one tenth of the volume of an immature spermatid Formation of the enzyme filled acrosome cap by the Golgi apparatus and its attachment to the nucleus Development of flagellum structures and their implantation to the nucleus. (14) The spermatids develop thus the configuration which enables them to leave the germinal epithelium during a complex process, called spermiation. In summary, the differentiation of spermatids may be divided into 8 steps, demonstrated in figure 5A. Normally, a large number of spermatids is malformed. Malformations may affect only the acrosome, the nucleus or flagellum or may be combined thus sometimes producing bizarrely abnormal spermatozoa. They are abortive germ cells. A large variety of malformed spermatids may develop: Malformations of the acrosome, absence of acrosome in cases of round-headed spermatids, disturbances of nuclear condensation, malformations of the flagellum, absence of parts of the flagellum, e.g. the middle piece, appearance of multinucleated spermatid giant cells and more (Figs. 5B, 5C, 6A, 6B, 6C, 6D) Spermiation The delivery of mature spermatids from the germinal epithelium (spermiation) is managed by the Sertoli cells. As a result of the complex cooperation of intermediate filaments and cytoplasmic tubules of the Sertoli cells spermatids are advanced to the border of the lumen of the seminiferous tubule. There the mature spermatids close their intercellular bridges, disconnect their contact to the germinal epithelium and become free cells, now called spermatozoa. (15) Smaller parts of the spermatids with cumulated RNA granules, a few mitochondria, lipid droplets and membranes are released, forming the so-called residual bodies. Most of them are incorporated and digested by the Sertoli cells. Characteristics of normal spermatogenesis on the basis of histological sections - Diameter of the seminiferous tubule 180 m at the minimum - Presence of A pale type-, A dark type-, B type-spermatogonia - Presence of primary and secondary spermatocytes - Differentiation of spermatids - Zones of spermiation - Score count of 8 at the minimum (see section - "Score count for the evaluation of spermatogenesis"). - Lumen of the seminiferous tubule - Normal lipid distribution in the Sertoli cell cytoplasm - Presence of stages of spermatogenesis - Formation of clones of germ cells

Thickness of the lamina propria of the seminiferous tubule of 8 m at the maximum - Normal structure and distribution of Leydig cells (16) Components of the intertubular space The intertubular space of the human testis contains the microvasculature, the endocrine Leydig cells, nerve fibres, macrophages, fibroblasts, further connective tissue cells (Co-cells) compartmentalizing in part this space and lymph vessels. Microvasculatureof the intertubular space is divided into Inter-Leydig cell-capillaries of the arterial side, Intramural capillaries in the peritubular wall of the seminiferous tubule Inter-Leydig cell capillaries of the venous side (Fig. 7A). The microvasculature accesses the seminiferous tubules and Leydig cells and permits distribution of endocrine and paracrine substances. Leydig cells ensheath parts of the microvasculature and deliver hormones into the vessels. Under pathological conditions the intima of capillaries may be thickened thereby narrowing the lumen. Another aspect is the apposition of outer layers of the capillary wall by additional connective tissue ground substance. In both cases the blood flow may be reduced. In consequence of this aspect of patchy arteriosclerosis focal degeneration of testicular tissue appears. (17) This finding is frequently observed in men with oligozoospermia of uncertain genesis. Leydig cells are prominent cells of the intertubular space. They constitute groups surrounding the capillaries. Leydig cells produce and secrete among others androgens, the male sex hormone, the most well known of which is testosterone. Testosterone activates the hypophyseal-testicular axis, the masculinization of the brain and sexual behaviuor, the initiation, processing and maintenance of spermatogenesis, the differentiation of the male genital organs and secondary sex characteristics. Recent investigations elucidated that the Leydig cells possess neuroendocrine properties in addition to their endocrine functions. There is evidence that Leydig cells express serotonin, catecholamine synthesizing enzymes, different antigens characteristic for nerve cells as well as neurohormones and their receptors, neuropeptides, cell adhesion molecules, components of the NO/cGMP-system, components of the renin/angiotensin system, neurofilament proteins, synaptic and storage vesicle proteins, and numerous growth factors and their receptors. Furthermore, Leydig cells possess antigens characteristic for glial cells such as astrocytes, oliogodendrocytes and Schwann cells. All these features characterize the Leydig cells as non-dividing, post-mitotic neuroendocrine cells with pluripotent properties. (18) Some of the neural antigens (e.g. substance P, NO, C-type natriuretic peptide, catecholamines, IGF-1, TGF-, PDGF) are involved in autocrine and/or paracrine regulation mechanisms of the testes such as testosterone production and release, maintaining of the hypothalamo-hypophyseal-gonadal axis, the local

communication between the somatic cells of the organ (Leydig cells, peritubular cells, Sertoli cells), the regulation of blood flow in the testicular blood vessels and the permeability of hormones and nutritive substances, as well as the contractility of seminiferous tubules and of the testicular capsule (tunica albuginea). Other substances discovered are seemingly rudiments that have been active during testis morphogenesis. The Leydig cells now are considered a part of the general neuroendocrine cell system. (19) Many different aspects of Leydig cell organization and function like degeneration, hyperplasia, or tumorous degeneration appear commonly along with disturbances of spermatogenesis. The number of Leydig cells does not necessarily correlate with hormone production. It has been shown by immunohistochemical investigations that in cases of an increased number of Leydig cells testosterone production takes place in few Leydig cells only. Fibroblasts Are randomly distributed in the intertubular space. In part they engulf groups of Leydig cells, capillaries and seminiferous tubules and represent compartmentalizing cells (Co-cells) (Fig. 7A). (20) Immunocytochemical investigations indicate that Co-cells express antigens characteristic not only for fibroblasts but also for glia cells. Larger bundles of nerve fibrescommonly follow the septula testis for innervation of blood vessels, but are also encountered in the intertubular space. They cross groups of Leydig cells and in part contact seminiferous tubules. The function of these fibres is still a matter of debate. (21) Macrophagesare a normal constituent of the intertubular space. Single cells are attached to the seminiferous tubules, Leydig cells or partly to blood vessels. Under conditions of inflammation or testicular cancer, the number of macrophages is increased. Morphologically different immigrant macrophages appear. Macrophages are able to enter the lumen of seminiferous tubules and to phagocytose spermatozoa. (22)Free lymphocytes are normally missing from the intertubular space of the human testis. Under special conditions, e.g. degeneration of seminiferous tubules, infections, allergic reactions and tumor cumulated free lymphocytes (infiltrates) are present. (23) Lymph vessels in the human testis are only found in the septula testis and very seldom in the intertubular space. (24)This organization differs completely from the condition in laboratory animals, e.g. rats, where lymphatics are a main component of the intertubular space. Kinetics of spermatogenesis Spermatogenesis commences during puberty and continues throughout life and until old age because of the inexhaustible stem cell reservoir. An abundance of germ cells are developed and delivered from the seminiferous tubules. The process of spermatogenesis is highly organized: Spermatogonia divide continuously, In part remaining spermatogonia,

In part giving rise to spermatogenesis. Originating from dividing spermatogonia, Cell groups migrate from the basal to the adluminal position of the germinal epithelium. Cell groups of different development are met in a section of a seminiferous tubule and contribute to the typical aspect of the germinal epithelium. (25) Six of these typical aspects were described in the human testis as "stages of spermatogenesis". In any given region of the germinal epithelium every 16 days the same typical aspects of germ cell groups appear. This space of time is called "cycle of the seminiferous epithelium". The development of an A type spermatogonium up to mature spermatids requires 4,6 cycles, e.g. 74 days. The mature spermatids delivered from the germinal epithelium as spermatozoa are transported through the epididymal duct system during additional 12 days. Therefore, 86 days at the minimum must be calculated for a complete spermatogenetic cycle from spermatogonium to mature spermatozoa. The products of spermatogenesis: spermatozoa Spermatozoa with their unique shape are suitable for the transport to the female gamete. For this reason the nucleus of the spermatozoon is condensed, covered by an acrosome for establishing contact to the female gamete and connected with a flagellum for progressive motility. The diameter of the head of spermatozoon is 45 m, the diameter of the flagellum is of 12 m and the length of the spermatozoon measures 60 m. The morphology of the human spermatozoon is depicted in figure 7B. Spermatozoa acquire their competence of motility during the transport troughout the epididymal ducts. Different processes of the maturation of membranes and surface coat substances take place. Principally, the motility of spermatozoa depends from normal development of the axoneme structures (e.g. microtubule doublets, dynein arms, etc.), the presence of mitochondrial sheath and the implantation of the flagellum at the nucleus by the both centrioles. Missing parts of the flagellar structures (e.g. dynein arms of the axoneme) give rise to immotility. Efficiency of spermatogenesis Spermatogenesis is a process of redundancy and of little efficiency in terms of quality management: Germ cell loss during spermatogenesis and the number of malformed spermatozoa in the ejaculate are extremely high. Calculating the potential spermatogenetic capacity of a testis with 100%, it must be realized that 75% of the developed germ cells are lost by apoptosis or degeneration. Only 25% of the germ cells reach the ejaculate and more than half of them are malformed. Therefore, only 12% of the spermatogenetic potential is available for reproduction.

In comparison to laboratory animals the spermatogenetic efficiency in man appears to be poor. (26) In this respect one value of interest is the mean elongate spermatid-Sertoli cell ratio being 34 for the human germinal epithelium, (versus 12 in rats). (27) Even under these conditions the daily rate of spermatozoa production in man is calculated as 34 millions per gram of testicular tissue. Based on these calculations a higher number of spermatozoa in the ejaculate should be expected than the rather low value of 20 millions of spermatozoa/ml ejaculate, as suggested by the WHO manual as normal value. (28) In recent years reports have been published, indicating a decline of spermatozoal concentrations in ejaculates of healthy males during the last decades. It is thought that interfering prenatal influences to the embryonal development of male gonads occur, e.g. by hormones and their metabolites in the drinking water and other nutriments of the mother. Regulation of spermatogenesis The process of spermatogenesis in the seminiferous tubules is maintained by different internal and external influences (29) Intrinsic regulation The Leydig cells in the intertubular space secrete testosterone and additional neuroendocrine substances and growth factors. These hormones, transmitters and growth factors are directed to neighbouring Leydig cells, to blood vessels, to the lamina propria of the seminiferous tubules and to Sertoli cells (Fig. 8). They are involved in maintenance of the trophic of Sertoli cells and the cells of peritubular tissue; they influence the contractility of myofibroblasts and in that way regulate the peristaltic movements of seminiferous tubules and the transport of spermatozoa. They also contribute to the regulation of blood flow in the intertubular microvasculature. Furthermore, different growth factors (IGF1, TGF, NGF) are delivered from Sertoli cells and several types of germ cells and take part in a complicate circle of regulation of cell functions and developmental processes of germ cells. All factors together represent an independent intratesticular regulation of spermatogenesis. This very intricate system has been investigated mainly in laboratory animals and is still less understood in human. (30) Extrinsic influences The local regulation of spermatogenesis in the testis requires the well known extratesticular stimuli provided by the hypothalamus and hypophysis. Pulsatile secretion of gonadotropin releasing hormone (GnRH) of the hypothalamus initiates the release of luteinizing hormone (LH) of the hypophysis. As a result of this stimulus Leydig cells produce testosterone. Testosterone influences not only spermatogenesis in the seminiferous tubules of the testis but is also distributed throughout the body and provides feedback to the hypophysis related to the secretory activity of Leydig cells. Stimulation of Sertoli cells by the pituitary follicle stimulating hormone (FSH) is necessary for the maturation of germ cells. The

Sertoli cells itself secrete inhibin in the feedback mechanism directed to the hypophysis. The extratesticular influences are a necessary basis for the function of intratesticular regulations. (31) Disturbances of spermatogenesis Proliferation and differentiation of the male germ cells and the intratesticular and extratesticular mechanisms of regulation of spermatogenesis can be disturbed at every level. This may occur as a result of environmental influences or may be due to diseases that directly or indirectly affect spermatogenesis. In addition, Different nutrive substances Therapeutics Drugs Hormones and their metabolites Different toxic substances X-radiation may reduce or destroy spermatogenesis. Finally, also a rather simple noxe as, Increased temperature reduces the spermatogenetic activity of the testis. Under these negative influences the testis answer rather monotonuous by reduction of spermatogenesis. This may be expressed. In the reduced number of mature spermatids, In malformation of spermatids, Missing spermiation, Disturbance of meiosis, Arrest of spermatogenesis at the stage of primary spermatocytes, Reduced multiplication Apoptosis of spermatogonia. If spermatogonia survive then spermatogenesis may be rescued. Otherwise spermatogenesis ceases and shadows of seminiferous tubules remain. Disturbances of spermatogenesis are evaluated in histological sections of testicular biopsies. (See page----Testicular Biopsy) Results of histological evaluation of testicular tissue are given in a score count:

(32) Score count for the evaluation of spermatogenesis Intact spermatogenesis: many mature spermatids and zones of spermiation Modest reduced spermatogenesis: reduced number of mature spermatids, a few zones of spermiation

Distinct reduced spermatogenesis: few mature spermatids, no spermiation Considerably reduced spermatogenesis: no mature spermatids, only immature spermatids, no spermiation Severely reduced spermatogenesis: only few immature spermatids, reduced height of germinal epithelium Arrest of spermatogenesis at the stage of primary spermatocytes: many spermatocytes border the lumen of the seminiferous tubule Arrest of spermatogenesis at the stage of primary spermatocytes: a few primary spermatocytes are present Arrest at the stage of spermatogonia: A type spermatogonia multiplicate but do not develop to maturing cells of spermatogenesis No germ cells, only Sertoli cells are present No germ cells, no Sertoli cells. The seminiferous tubule is replaced by connective tissue ground substance (shadow of tubule)

(33) Understanding spermatogenesis under the aspect of assisted reproduction Nowadays availble methods of assisted reproduction are founded in basic knowledge of spermatogenesis. By means of specialized techniques of extraction of spermatozoa from testicular tissue (TESE) in combination with intraovocytoplasmatic injection of spermatozoa (ICSI) pregnancies could be induced. (34) Even in deleterious cases of male infertility (zoosperm in the ejaculate, high levels of FSH) in more than 50%, after using Ayurvedic treatments, male gametes could be detected and used for fertilization. The identification of single mature spermatids and spermatozoa is ascertained by means of detailed morphological techniques. Finally, unrelated these sufficient results it is remarkable that in 0.8 % of the cases under study early testis cancer could be revealed.