Anatomy and embryology of male and female reproductive systems

Germ cell specification

Three weeks after fertilization, from the proximal epiblast-derived mesodermal portion located near the extraembryonic endoderm—at the posterior part of the primitive streak—arises a group of cells (i.e., at least six in number) that display unique qualities from their surrounding tissues (i.e., larger size, clearer cytoplasm, and fewer organelles) and will later carry on their role as the PGCs. This process has been termed “specification” and is influenced by a number of factors—that belong to the transforming growth factor-β ( TGF-β ) superfamily—produced by extraembryonic ectoderm although the cascade response of the cells depends on the expression of other factors like the wingless-type MMTV integration site member 3 ( WNT3 ) (i.e., in this case to specifically respond to the bone morphogenetic proteins ( BMP ) of the TGF-β family). Genes like PR-domain containing-1 (i.e., Prdm1 or Blimp1 as previously called), Prdm14 , and transcription factor AP-2 gamma ( Tfap2c ) are downstream activation targets that should be properly expressed for a successful specification to take place since—for example— Prdm1 prevents the expression of genes that can pull the germ cell toward the somatic cell-line development. Rodriguez and colleagues provide an excellent discussion of growth factor and genes related to sex differentiation and development that can be found in their published work.

Germ cell migration and the concurrent genital ridge development ( Fig. 1.2 )

For the next 1–2 weeks post specification, PGCs acquire changes in their morphology (i.e., pseudopodia) in order to carry out active amoeboid movement and successfully migrate to the hindgut and then to the genital ridge (i.e., and into the developing sex cords). Their path goes through the dorsal mesentery and the journey seems to require the influence of various genes (i.e., homeobox protein Lim-1 ( LHX1 or Lim1 ) for proper hindgut localization and WNT family member 5A ( WNT5A ) for the PGCs to receive a proper migration cue; the mutation of the latter can partly explain findings in Robinow syndrome), the action of ligand/receptor pathways (i.e., stromal cell-derived factor 1 (Sdf1 or C–X–C motif chemokine 12 (CXCL12)/CXC receptor 4 (CXCR4) pathway), an optimum collagen-I deposition in the extracellular matrix (i.e., induced by TGF-β through the activin receptor-like kinase 5 (alk5)), and the help of autonomic nerve fibers migrating along with them to the same destination.

The development of the genital ridge commences and proceeds in conjunction with the migration. The urogenital ridge arises as a dorsal body wall thickening that harbors populations from both the primitive mesoderm and the coelomic epithelium (viz., adrenogenital primordium). The central portion—the mesonephros—of the urogenital ridge gives rise to the gonadal ridge along its ventromedial surface—which starts bulging after germ cells infiltrate it to form the primordial indifferent gonad by the end of the 5th week of intrauterine life. Certain gene mutations can affect the development of genital ridge (i.e., and consequently risk the lack of the development of the gonads themselves in addition to some malfunctions in the adrenal and renal systems) and they include Wilms’ tumor 1 ( Wt1 ), steroidogenic factor-1 ( SF1 ), Lhx1 , Lhx9 , empty spiracles homeobox 2 ( Emx2 ), and GATA-binding protein 4 ( GATA4 ).

The indifferent stage, the bipotential gonad, and the gonadal differentiation

Sustainment of the morphological development of the gonads requires the presence and the influence of germ cells only in the case of the ovary (i.e., normal testicular development continues in their absence) ; however, both the cortex and the medulla of the indifferent gonads change according to the genotype of the fetus (i.e., cortex develop and medulla regresses in the case of the XX genotype and the medullary sex cords proliferate toward seminiferous tubules formation in the case of the XY genotype). Interestingly, PGCs differentiate into male or female gametes according to the cellular environment they are placed in. For instance, female genotypic PGCs become spermatogonia when they are placed in gonads with testicular somatic cell environment (i.e., of Sertoli cell lineage) while those migrating to aberrant locations give rise to oogonia—that degenerate afterward—irrespective of their sexual genotype (i.e., though rarely they form teratomas in certain locations as the mediastinum). Assumptions about the sexual identity of the gonad grow stronger as intrauterine life progresses over the weeks depending on a number of clues—which when working toward an assumption of the presence of an ovary—that include the following: lack of testicular development beyond the 7th week; the presence of oogonial meiosis around the time of the 8th week; and visualization of primordial follicles by the 16th week.

Gonadal differentiation seems to rely on intricate genetic interactions between multiple upstream and downstream factors ( Fig. 1.3 ), many of which are yet to be fully elucidated. The long-held belief that the female differentiation pathway is the default one in the absence of male factors’ influences has recently started to be challenged. Consequently, for example, and in the case of female gonad determination and the hypotheses behind it, two possibilities are now entertained for the directions factor takes to achieve the determination: repression of somatic testis-inducing genes in addition to the activation of ovary stimulating ones as one possibility; downregulation of the same male genes while removing the repression on ovary stimulating ones as an alternate possibility. It is worthy of note that no definite hypothetical model has been adopted yet even when the knowledge about possible factors keeps-on expanding.

At about the 7th week and under the influence of the autosomal gene SRY (sex-determining region Y)-box 9 ( SOX9 ) (i.e., which gets inhibited mainly by β-catenin or catenin beta-1 gene ( CTNNB1 ) in the case of ovary determination pathway and is upregulated by the SRY gene), Sertoli cells differentiate, proliferate, and surround the arriving PGCs to form primitive sex cords while protecting the PGCs from entering meiosis prematurely (i.e., protection is vital for commitment to spermatogenesis and is mediated by degrading retinoic acid—and possibly other products—by the cytochrome p450 enzyme (Cyp26b1); mitotic arrest continues until puberty). Sertoli cells promote differentiation of peritubular myoid cells and—by the 8th week—Leydig cells (i.e., both arising from primitive interstitial cells) by secreting anti-Müllerian hormone (AMH) as a paracrine factor. SRY and other factors (i.e., transmembrane ligand ephrin-B2) influence to proper vascularization of the testes—which is characterized by early distinct arterial specification and is necessary for further transportation of testicular hormones. From the second trimester thereafter, Sertoli cells double in number fortnightly (i.e., coupled by a parallel increase in numbers of germ and Leydig cells) resulting in a rapid increase in testicular sizes. It is evident that Y-chromosome genes are necessary to maintain spermatogenesis while having extra X-chromosomes (i.e., as is the case with Klinefelter’s syndrome) can result in compromised testicular development and size.

An alternate developmental path is generally followed in the case of the ovary and differs from that of the testis in the following points:

• Failure of migration of PGCs results in a degenerated fibrous streak being formed (i.e., Turner syndrome). After their successful arrival and infiltration of the secondary cortical sex cords, the medulla starts to regress and the ovaries start to become distinguishable by the 6–8th weeks. PGCs undergo vigorous mitotic duplication during that time (i.e., reaching 600,000 in number as oogonia by the 8th week); subsequently, meiosis (i.e., which is facilitated by the action of retinoic acid and possibly nonretinoic acid factors), oogonial atresia, and the ongoing mitosis regulate their number until they reach a peak of at least 6 million cells by mid-gestation—at the time of formation of the first primordial follicle and breakdown of oocyte cell nests—and then decline afterward (i.e., cells entering meiosis freeze their progress at the meiosis-I phase by the 17th week to resume meiosis later in puberty). By the 7th month, mitosis is terminated and oogonial atresia is replaced by follicular atresia which withers their number to only 700,000 primordial follicles at birth (i.e., and 300,000 by the time of puberty). Fig. 1.4 provides more details about oogenesis throughout the intrauterine period.

  

• Pregranulosa cells lack SRY expression that allows SOX9 to be antagonized properly and—thus—the commitment to granulosa cell lineage can occur under the influence of the WNT4/β-catenin pathway.

• The forkhead box 2 ( Foxl2 ) gene maintains such differentiation by continuous suppression of SOX9 .

• Nuclear receptor subfamily 0 group B member 1 ( NR0B1 ) gene (formerly called dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 ( DAX1 )) is a dosage-sensitive gene that needs two copies to antagonize SOX9 and SF1 . Conversely, as suggested by its formal name, the absence of NR0B1 gene (i.e., low dose) can potentially result in testicular dysgenesis.

• Theca cells are the Leydig cells’ female counterparts and developed from the interstitial cells under the influence of WNT4 and Foxl2 genes and in the absence of the effect of high levels of testosterone.

• Vascularization of the ovary follows the classic angiogenic route rather than recruiting endothelial cells by breaking down mesonephric vessels.

Gonadal relocation and descent ( Fig. 1.5 )

Testicular migration—and descent—begins around the 12th week of intrauterine life, goes through two major phases (viz., transabdominal and inguinoscrotal phases), and ends with the successful intra-scrotal positioning by the 32nd gestational week. In addition to the increase in the intraabdominal pressure and the passive change in position that occurs as a result of elongation of the trunk and enlargement of the pelvis (i.e., both phenomena are common to both sexes), the descent is aided in males by the secretory products of Leydig cells (including testosterone and insulin-like factor 3 (Insl3) and anti-Müllerian hormone (AMH)) that result—first—in the simultaneous regression of the craniosuspensory ligament and differentiation of the gubernaculum (i.e., to develop muscular tissue) and then the contraction of the latter to guide the testes through the inguinal canal (i.e., that contracts around the spermatic cord afterward preventing inguinal hernia) and into the scrotum—a process that the genitofemoral nerve is hypothesized to mediate through the action of calcitonin gene-related peptide (CGRP). Mutations in the INSL3 gene or its receptor gene (viz., relaxin/insulin-like family peptide receptor 2 [ RXFP2 ], aka leucine-rich repeat-containing G protein-coupled receptor 8 [ LGR8 ] or G protein-coupled receptor affecting testis descent [ GREAT ]), deficient Insl3 production, or fetal androgen insufficiency/resistance are associated with cryptorchidism in humans.

In females, the craniosuspensory ligament remains intact (i.e., which later gives rise to the ovarian suspensory ligament) while the gubernaculum remains undifferentiated (i.e., which later gives rise to the round ligament of the ovary [superior part] and that of the uterus [inferior part] which is anchored to both labia majora). Therefore, the ovaries only undergo a caudal shift to become positioned lateral to the kidneys.

Sexual duct differentiation ( Fig. 1.6 )

At week 4 of gestation, the indifferent stage of sexual duct differentiation begins as the mesonephric duct (aka Wolffian duct) forms from the lateral nephrotomes (i.e., intermediate mesoderm segments), followed 2 weeks later by the emergence of the paramesonephric duct (aka Müllerian duct) as an invagination of the urogenital ridge’s surface epithelium. After another 2 weeks, the testosterone secreted by the Leydig cells induces the differentiation of the Wolffian ducts into the epididymides (cranial segment), vasa deferentia, and seminal vesicles (lower segment) over the succeeding 4 weeks (i.e., dihydrotestosterone (DHT) does not influence the process as evident from the lack of 5α-reductase 2 enzyme expression in Wolffian ducts during differentiation). A simultaneous process of defeminization takes place where the Müllerian ducts regress under the effect of the AMH to completely disappear by the 11th week (i.e., AMH—or Müllerian inhibitory substance (MIS)—produced by Sertoli cells and the mechanism is mainly an apoptotic one). Both hormones might be primarily acting in a paracrine manner since unilateral testicular absence leads to ipsilateral persistence of the Müllerian structures and residual development of Wolffian ones ; inactivating mutations of AMH gene or AMH type II receptor defects can also negatively impact the functions of AMH (i.e., persistent Müllerian duct syndrome (PMDS) while overexpression in females leads to blind vagina and the absence of oviducts. Conversely in normal 46,XX females, the absence of the AMH allows the Müllerian ducts to continue their differentiation into fallopian tubes (cranial portions), uterus, cervix, and the upper third of the vagina (i.e., the lower portion originates from the urogenital sinus) while the absence of the testosterone prevents the differentiation of the Wolffian ducts (i.e., thus only nonfunctioning remnants can persist).

Some genetic defects can potentially result in the complete absence of reproductive tracts including the null mutation of Emx2 , Lim-1 , and paired box 2 ( Pax2 ) genes; WNT4 and WNT7A mutations are accompanied with the masculinization of reproductive tracts in females in the case of the former and with Müllerian regression failure in males (causing obstructive sterility)—and Müllerian defective differentiation in females—in the case of the latter. Homeobox-containing genes ( HOX ) are very important in the specification of various male and female tract parts and are expressed from top downward with increasing numbering (i.e., Hoxd10–13 or its Hoxa paralogues) such that mutations of—for example— Hoxa10 leads to the transformation of the cranial uterine part into a fallopian tube in females and ductus deferens to epididymis in males. In humans, HOXA13 mutation leads to the hand–foot–genital syndrome (aka Guttmacher syndrome) where there are Müllerian defects in females and hypospadias in males (i.e., in addition to limb abnormalities).

External genitalia and other accessory glands differentiation

The external genitalia of both sexes develop indifferently until the 8th week of gestation (i.e., the start of testosterone secretion); before then, an initial elevation called the genital tubercle is formed—at the cloacal membrane’s cranial end and possibly guided by fibroblast growth factor 8 ( Fgf8 ) gene—by the 4th week (i.e., primordial penis or clitoris), and then the urethral (medial) and labioscrotal (lateral) folds (i.e., primordial labia minora/penile foreskin and labia majora/scrotum, respectively) flank the urogenital groove by the 6th week and the indifferent stage appears largely hormonal-independent.

The presence of DHT (i.e., converted by the action of 5α-reductase enzyme on testosterone) and its action on androgen receptor (AR) in the target tissues (i.e., external genitalia and some accessory glands like the prostate) is the deciding factor for masculinization of them (i.e., in the case of deficiency of DHT—or in the case of resistance like in androgen insensitivity syndrome—the external genitalia develop down the female pathway). By the 12th week of gestation, those going down the path of masculinization complete the process of male sexual differentiation of the external genitalia (i.e., genital tubercle elongation as penis, urethral folds ventral fusion as penile urethra, and labioscrotal folds midline fusion as scrotum) and the prostate (i.e., develops from the urogenital sinus after the vesicovaginal septum formation is inhibited)—penile enlargement, however, continues further in the third trimester (i.e., which seems quite incomplete in cases of fetal hypopituitarism—or luteinizing hormone receptor β (LHβ) mutations—since the androgen production is less dependent on the maternal chorionic gonadotropin (CG) stimulation in the third trimester) and the whole penile enlargement appears to be under the regulation of HOX , FGF , and Sonic hedgehog ( Shh ) genes. In the absence of proper androgen stimulation (i.e., female, androgen deficiency, or androgen resistance), the external genitalia continue down the female pathway and also complete the differentiation by the 12th week as follows: genital tubercle slightly enlarges as clitoris, urogenital sinus remains open, vesicovaginal septum forms, urethral folds become labia minora, and labioscrotal folds become labia majora (i.e., minor labial fusions lead to the formation of mons pubis (anteriorly) and posterior commissure (posteriorly)). Urethral and urogenital sinus outgrowths produce urethral/para-urethral glands and great vestibular glands, respectively. Table 1.2 illustrates homologous parts of the urogenital system of both genders.

Testes and spermatogenesis

The male gonads (testes) are the most important component of the male reproductive system since it is the sole source of the gametes and the main source of male sex hormone (testosterone). As per the currently available literature, each testis is an oval-shaped firm organ with measurements ranging from 3.7 to 5.1 cm in terms of length, 2 to 3.1 cm in terms of width, and 3 to 5.2 cm in the anteroposterior diameter; measurements determined clinically are useful for estimating volume (i.e., by incorporating them in equations) and can be used as cutoffs to help diagnose certain syndromes (i.e., < 3.5 cm longest diameter Klinefelter’s syndrome ). Additionally, each normally possesses a weight that ranges from 12 to 26 g and a volume that ranges from 14.8 to 28 mL (i.e., clinically or a US measured range of 10–17 mL ).

For spermatogenesis to proceed properly, the temperature of the testes has to be held at a level 3–4°C lower than the cores and this is normally facilitated by several factors: their position in the scrotum outside the body (i.e., which is continuously corrected by the cremasteric muscle according to testicular and scrotal temperatures), the special characteristics of the scrotal skin (see later), and the pampiniform venous plexus maintained countercurrent exchange mechanism (i.e., by surrounding the testicular artery with their colder blood). Conditions that increase such temperature result in impaired spermatogenesis (i.e., cryptorchidism and frequent hot baths; the latter can cause a temporary effect with a recovery of at least 3 months).

The testes usually lie obliquely suspended by the spermatic cords in the scrotum and buried inside three layers of coverings (i.e., ordered from outside inward): a peritoneum-like tunica vaginalis (i.e., covering all but the testicular posterior aspect; it’s the extension of the processus vaginalis whose incomplete closure predisposes to indirect inguinal hernia and hydroceles), the fibrous tunica albuginea, (i.e., which creates the mediastinum testis) and the vascular tunica vasculosa (i.e., which is in-line with all surfaces of the testis and tunica albuginea’s septations going into it). Fig. 1.7 provides an illustration of the testes and their surrounding relationships.