Although multiple genes play a role in testicular differentiation, the two genes of prime importance are NR5A1 and WT-1 (Wilms tumor gene). NR5A1 is located on chromosome 9q33.3 and comprises seven exons with a gene product known as SF-1 (steroidogenic factor 1). SF-1 is initially recovered from the Sertoli cells of the sex cords, but it is localized to the Leydig cells during later developmental stages.¹ It enhances the expression of anti-müllerian hormone (AMH) and plays a role in regulation of the AMH gene. A female phenotype is found in 46XY subjects with a heterozygous deletion of NR5A1 along with other features including adrenal failure in the first months after birth, persistence of normal müllerian structures and maldeveloped gonads comprising poorly differentiated tubules with abundant connective tissue stroma. The neonatal phenotype is not a reliable predictor of virilization at puberty. Male gender assignment in poorly virilized cases at birth may allow spontaneous puberty without signs of hypogonadotropic hypogonadism, and possibly fertility. Patients with SF-1 mutations are at increased risk for malignant germ cell tumors. Early orchidopexy and germ cell tumor screening are mandated in the presence of preserved gonads. In cases where premalignant and/or malignant changes are identified, gonadectomy with or without possible irradiation constitutes the modality of treatment.² Adrenal failure is the sole presenting feature in patients with 46XX since SF-1 does not influence ovarian development.³ These disorders aid in highlighting the significant position of NR5A1 expressed in the primitive urogenital ridge, which differentiates into the gonads and adrenal glands.

WT1 is located on chromosome 11p13 and contains 10 exons with alternative splicing sites in introns 5 and 9. The splicing of intron 9 can result in KTS+ (three amino acids viz. lysine, threonine, and serine) or KTS- isoforms. Normal gene expression mandates a proper balance of both isoforms. The WT1 gene is predominantly expressed in the kidneys and gonads. It is responsible for stromal-epithelial transition and inhibition of genes encoding proliferative factors for epithelial differentiation and simultaneous activation of genes enhancing the process. A host of phenotypic alterations are observed with WT1 gene anomalies.⁴ Loss of the KTS+ isoform gives rise to Frasier syndrome characterized by 46XY gonadal dysgenesis, absence of Wilms tumor, and renal disease of late-onset type.⁵ Missense heterozygous mutations are manifested as Denys-Drash syndrome with partial or complete 46XY gonadal dysgenesis, Wilms tumor, and earlyonset renal disease with diffuse mesangial sclerosis.⁶ WT1 deletions are linked to an increased propensity to develop Wilms tumor and variable genitourinary system manifestations. Other genes identified in the formation of kidneys and gonads are LIM1 and FGF-9 (fibroblast growth factor 9).

The signal for gonadal differentiation is initiated by the SRY gene on the sex-determining region of chromosome Y (Yp11.3) otherwise known as testis determining factor gene.⁷ It is this gene that is responsible for production of the AMH, differentiation of Sertoli cell precursors and germ cells, and downstream gene regulation.⁸ The pathway of testicular differentiation is complex and involves activation and inhibition of both autosomal and sex chromosomal genes. The SRY gene has been demonstrated in the nuclei of germ cells and Sertoli cells. It contains a single exon encoding a 204-amino acid protein of the central part that is responsible for encoding a DNA-binding domain referred to as high mobility group (HMG). SRY also regulates steroid hormonal expression and interacts with the AMH promoter gene.⁹ Mutations of SRY give rise to pure gonadal dysgenesis (Swyer syndrome) or true hermaphroditism. Although all affected cases possess male external genitalia and testes, they have no müllerian structures and azoospermia. The karyotype of patients with the male phenotype without the Y chromosome is 46XX SRY+ in 80% cases and 46XX SRY- in 20% cases. SOX9 duplication may also be present in some instances.¹⁰

Several other genes encode associated transcription factors and play a role in gonadal differentiation including DAX-1, SOX-8, SOX-9, LHX-9, LIM-1, and DMRT-1. DAX-1 (dosage-sensitive sex reversal) gene is situated on X chromosome and is part of the pathway for development of ovaries, testes, and adrenal glands. It is inhibited by SRY during testicular differentiation and activated during ovarian differentiation. DAX mutations are associated with decreased levels of gene expression causing nondevelopment of the adrenal cortex and hypogonadotrophic hypogonadism with normal testicular development.¹¹ Human DAX1 duplications result in dosage-sensitive sex reversal (DSS) subsequent to which individuals with a chromosomal XY pattern can develop as females due to gonadal dysgenesis. The exact mechanism of DSS-adrenal hypoplasia congenita on X, gene 1 (DAX1) action in the fetal testis is albeit unknown. It has been demonstrated that in fetal testes from XY Dax1-overexpressing transgenic mice, the expression of the key testis-promoting gene sex-determining region on Y (SRY)-box-9 (Sox9) is reduced. Also, in XY Sox9 heterozygotes, in which testis development is usually normal, Dax1 overexpression results in ovotestes, thereby indicating a DAX1-SOX9 antagonism. The ovarian portion of the XY ovotestes in a recent study was characterized by expression of the granulosa cell marker, forkhead box-L2, with complete loss of the Sertoli cell markers, SOX9 and AMH, and the Leydig cell marker CYP17A1. However, the expression of SRY and SF-1, two key transcriptional regulators of Sox9, was retained in the ovarian portion of the XY ovotestes. Dax1 overexpression reduced activation of TES, the testis enhancer of Sox9, indicating that DAX1 might repress Sox9 expression via TES in reporter mice. Increasing levels of DAX1 antagonized SF-1-, SF-1/SRY-, and SF-1/SOX9-mediated activation of TES in cultured cells, as a result of reduced binding of SF-1 to TES, thus providing a possible mechanism for DSS.¹²

SOX-8 and SOX-9 (SRYY box 8 and 9) are linked to autosomal genes. SOX9 is located on chromosome 17q24. 3q25.1 and is expressed after SRY expression in pre-Sertoli cells.¹³ The protein encoded by this gene recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins. It acts during chondrocyte differentiation and, with SF-1, regulates transcription of the AMH gene. Functional allelic losses lead to the skeletal malformation syndrome (campomelic dysplasia), frequently with 46XY constitution with female phenotype.¹³ Duplication of SOX-9 results in 46XX patients with male phenotype.¹⁴ SOX-8 is another gene involved in AMH regulation and interacts with SF-1 through protein-protein interactions. It has been demonstrated experimentally that SOX-9 dysfunction leads to SOX-8 expression as a replacement through a feedback process.¹⁵

Deletions in chromosome 9p¹⁶ and 10q¹⁷ result in expression of a female phenotype in 46XY genotype cases. Deletions of chromosome 9p are also associated with hydronephrosis, facial malformations, and delayed development. In 46XY females, deletions of two genes (DMRT1 and DMRT2) are located on chromosome 9p24.3. Chromosome 10q deletions are associated with genital malformations, mental retardation, and other systemic manifestations.

Multiple hormones are involved in the development of the male genital system at various stages, including AMH, testosterone, dihydrotestosterone (DHT) and the pituitary gland hormones, follicle-stimulating hormone (FSH), and the luteinizing hormone (LH).

AMH (also known as müllerian inhibitory substance, MIS) is a glycoprotein consisting of two identical 72-kDa subunits linked by disulfide bonds that is secreted by Sertoli cells in males and granulosa cells in females.¹⁸ Its expression is regulated by SF-1, which is a transcriptional regulator of many steroid genes.¹⁹ AMH is a member of the TGF-β family encoded by a 2.75-kb gene situated on 19p13.2. The amount of AMH secreted is inversely proportional to degree of Sertoli cell maturation. It can be detected during the 8th to 9th week of gestation, and its concentration rises in the second trimester and decreases significantly in the third trimester.²⁰ Although its level rises upon birth and AMH is detectable during childhood, its levels decline to undetectable with the onset of puberty when it is negatively regulated by androgens.²¹

The target sites of AMH include the genital tract, testis, and surrounding structures with AMH causing involution of the ipsilateral müllerian duct beginning from the caudal pole with rapid progression upward. It regulates SRY expression being expressed around the same time frame. The tunica albuginea forms through mesenchymal insertion between primitive sex cords and coelomic epithelium and its development is promoted by AMH.²² This hormone presents a barrier to spermatogonia entering meiosis.²³ Of note is the role played by AMH in the development of fetal lungs.²⁴

Testosterone synthesis commences during the 8th week of gestation and is synthesized by Leydig cells, which appear during the 8th week of gestation and constitute approximately 50% of the testicular volume by the 16th week.²⁵ However, the secretion of testosterone is regulated by hCG and LH levels. hCG levels are at their peak during the 11th to 18th weeks and fall to significantly lower levels after this duration. This period of hCG-dependent testosterone secretion is crucial in terms of genital differentiation. Wolffian duct differentiation occurs when testosterone is secreted by the testis on each side and leads to differentiation of epididymis, ductus deferens, and seminal vesicle. Defects in androgen synthesis are evidenced as cryptorchidism and incomplete masculinization.

The enzyme 5α-reductase acts on testosterone to produce DHT, which in turn is responsible for differentiation of the prostate along with external genitalia, male urethra, scrotum, and penis. The midline fusion of labioscrotal folds (day 70) to form the scrotum with the midline raphe is induced by DHT.
The penile urethra is formed by fusion of the urethral folds (day 74), and the genital tubercle subsequently enlarges to form the glans penis. The terminal urethra develops from an invagination of the glans tip. The prostate, urinary bladder, and prostatic urethra are formed from the urogenital sinus.²⁶

The roles played by FSH and LH gain importance toward the last weeks of gestation. LH levels start rising in fetal circulation during the 10th week and peak by the 18th week after which they decline gradually until birth. LH regulates fetal androgen production in the second and third trimesters. FSH is responsible for Sertoli cell mitogenic activity, which peaks at the time of delivery.²⁷

In their initial stages, germ cells acquire diverse evolutionary morphologic appearances. Three types of germ cells have been identified, and some authors²⁸ have proposed that these should be known as gonocytes (OCT4 positive, c-kit positive), intermediate germ cells (OCT4 low expression/negative, c-kit negative), and prespermatogonia (OCT4 negative, c-kit negative). In the first trimester, most germ cells have a gonocyte phenotype; however, from the 18th week of gestation, prespermatogonia are the most abundant cell type (Fig. 11-1). These data provide evidence for the functional differentiation of human testicular germ cells during the second trimester of pregnancy and argue against these germ cells being considered a homogeneous population.

From birth until puberty, the testicles develop continuously, but some phenomena permit this period to be subdivided into the following three phases:

When the testicle is totally developed, it has a supporting structure—the albuginea—that surrounds the testis like an external capsule. Between the thickened mediastinum testis
area and the surface of the organ are fibrous septa that project toward the interior and divide the testicular parenchyma into about 250 segments or lobules. The albuginea has three layers; from outside to inside they include the external mesothelial layer (tunica vaginalis testicular); the medial, relatively acellular layer with fibroblasts, myocytes, and nerve fibers (tunica albuginea); and the internal vascular layer (tunica vasculosa).³⁰

The seminiferous tubules occupy 70% to 75% of the testicular volume. Each seminiferous tubule has a closed-loop structure with intercommunication between the arms of the loop. The size varies according to the section angle. In the cross sections of the tubules in an adult, the average diameter is about 180 μm. Each seminiferous tubule is composed of a tubular wall, Sertoli cells, and germ cells (Table 11-1).

The tubular wall comprises five layers; they include the basement membrane (periodic acid-Schiff positive), the internal acellular layer, the internal myofibroblastic layer, the external acellular layer, and the external fibroblastic layer from the inner to the outer aspect. The innermost strata have contraction functions, apparently useful for intratubular motility, and thus mobilize the spermatozoids toward the rete testis or network of canals at the termination of the straight seminiferous tubules in the mediastinum testis.³¹ The elastic fibers appear in puberty, are located more externally, and may be lacking in dysgenetic testicles.³²

The Sertoli cells (10.2 ± 2 per tubule) have abundant cytoplasm with triangular nuclei and prominent nucleoli that extend from the basal membrane to the tubular lumen. Sertoli cells express vimentin and have the greatest metabolic activity of all cells in the entire tubule since they induce the process of differentiation and the maturation of spermatocytes, regulate the maturation of the Leydig cells, secrete inhibin, and produce tubular fluid.³³ These cells have strong desmosomal junctions in their lower portions and weak ones in their upper portions.³⁴ Sertoli cells synthesize and secrete a large variety of factors: proteins, cytokines, growth factors, opioids, steroids, prostaglandins that explain the presence of endoplasmic reticulum smooth and rough type and a prominent Golgi apparatus.

The germ cells progressively mature from spermatogonia to their final mature forms within 70 to 74 days. From the morphologic point of view, 13 distinct types of germ cells can be identified, but at the typical light microscopic level, only the following principal levels of maturation are recognized (Fig. 11-3):

The order of these germ cells in the tubule is apparently irregular, but for some time it has been recorded that there are six distinct stages,³⁵ a consequence of a helicoidal ordering throughout the tubule. These stages of spermatogenesis are defined as a characteristic association of germ cells representing several waves of spermatogenesis that occur simultaneously within the seminiferous tubules. Each stage has a specific duration, and the groups always occur in the same order, so that once the complete cycle is finalized it begins again.³⁶ During the maturation process,
from spermatogonium to mature spermatids, all the cells belonging to the same clone are interconnected.³⁷ Stage I is composed of spermatogonia, pachytene primary spermatocytes, and round and elongating spermatids. Stage II includes spermatogonia, pachytene primary spermatocytes, round and elongated spermatids, and residual bodies derived from spermatid cytoplasm within Sertoli cells. Stage III is characterized by the beginning of spermatid nuclear condensation and the entrance of type B spermatogonia into meiosis. Stages IV and V comprise pachytene primary spermatocytes, and can be differentiated by the presence of leptotene and zygotene primary spermatocytes. In Stage V, the secondary spermatocytes undergo a second meiotic division after a very short interphase.

In the interstitium, connective tissue collagen fibers with contractile capacity are present. The Leydig cells are the most important component, making up to 5% to 12% of the testicular volume.³⁸ They are polygonal and have PAS-positive, eosinophilic cytoplasm with a central nucleus and prominent nucleolus, grouped in small nests (1 or 2 nest per tubule or 5 ± 0.2 cells per tubule) around the capillaries of the intertubular space. The fetal Leydig cells produce testosterone; immature Leydig cells produce 3α and 17β-diol and adult Leydig cells steroids. Characteristically, some eosinophilic crystalline structures can be found in the cytoplasm (Reinke crystalloids); these are probably subunits of globular proteins whose functional meaning is not known³⁹ (Fig. 11-4). Other cellular elements of the interstitium are the macrophages that secrete interleukin 1, which stimulates the proliferation of the germ cells, and the mast cells.

The adult testis measures approximately 4 to 5 cm × 3.5 cm × 3 cm and weighs 15 to 19 g with the right usually being 10% heavier that the left. The external surface is covered by a capsule (tunica albuginea), which is a smooth and homogeneous layer measuring 400 to 459 μm in thickness in the adult. The tunica albuginea is composed of three layers. The outermost layer comprises of dense connective tissue and is lined by mesothelium. The middle layer represents less dense fibrous tissue. The inner most layer is rich vascular connective tissue. From this tunica albuginea layer, numerous fibrous septa emerge to divide the testicular parenchyma into approximately 250 lobules, and meet in a solid posterior area at the hilum of the testis, near the epididymis, which is called the mediastinum of the testis.

Each lobule of the testis contains one to four seminiferous tubules. Every tubule has a diameter of 180 μm and a total average length of 540 m. The tubules are a convoluted structure with numerous communications between the arms of the loop. Each arm of the loop empties into the mediastinum.

The mediastinum contains the rete testis, a connecting structure between the seminiferous tubules with the ductuli efferentes in the epididymis. There are around 1,500 units of seminiferous tubules to the rete. It is divided into three parts: the septal portion with the tubulae reti, which are short tubules 0.5 to 1.0 mm in length that connect the two ends of the seminiferous tubules to the mediastinal part with the tunical rete, which is a cavernous network of interconnecting channels between tubular reti and the extratesticular part with the bullae retis, which are vesicular channels measuring 3 mm in width that anastomose together to form the ductuli efferentes. In the mediastinal and external rete testis parts, fibrous columns or strands covered by epithelium called chordae retis are present to connect the different walls.

The rete testis epithelium is composed of flattened cells interspersed with small areas of columnar cells. Both cell types have single centrally located cilia and numerous microvilli on their free surfaces. These cells rest on a basal lamina surrounded by a layer of myofibroblasts and external layer of fibroblasts and collagen and elastic fibers.

Apart from the connection between the testis and the epididymis, the rete testis produces a pressure gradient internally for reabsorption of protein and potassium from tubular fluid.

The testis is supplied by the testicular artery, which arises from the abdominal aorta. In the spermatic cord, the testicular artery gives multiple branches that run along the interlobular septa of the testis. These centripetal arteries lead to the mediastinum testis and give off branches called centrifugal arteries.

The inner two-thirds of the testicular parenchyma are drained by veins that follow the interlobular septa to the mediastinum (centripetal veins). The outer third is drained by veins that lead to the tunica albuginea (centrifugal veins). Both centripetal and centrifugal veins join to form the pampiniform plexus, which drains the testis via the spermatic cord.

Lymphatic vessels are poorly developed in the testis and limited to the tunica vasculosa and interlobular septa where they accompany arterioles and venules.

Congenital disorders in number and size constitute one of the less frequently seen groups of testicular anomalies.

Some congenital anomalies can be classified by their locations outside the normal path of testicular descent (ectopia) or in the path of descent (undescended testes). The location
in the superficial inguinal pouch is considered to be ectopia by some researchers or to be cryptorchidism by others.