Development of the Murine Reproductive System

Overview

In the genital system, the first evidence of a genital (or gonadal) ridge is seen at about 10 dpc. This arises as a distinct, rostro-caudally elongated mound of tissue that develops on the medial aspect of the mesonephros. At this stage, and for approximately two days, it is not possible either histologically or morphologically to recognise whether the future gonad is destined to give rise to a testis, or to an ovary. For this reason, this period is referred to as the so-called “indifferent” gonad stage. Within a relatively short period of time, however, by about TS19 (11.5 dpc), there is considerable evidence of primordial germ cell colonization of the indifferent gonads. This is most readily seen if appropriate histochemical staining is employed, for example, to demonstrate the presence of high levels of intracellular alkaline phosphatase enzyme activity within these cells. It should be noted, however, that similar histochemical staining is also noted elsewhere in the embryo at this time, in regions that are unrelated to the presence of the primordial germ cells. This activity is also seen, for example, in the ventral part of the neural tube and in the apical ectodermal ridges of the developing limb-buds.

Despite the presence of the primordial germ cells in the developing gonads, there is still no clear evidence seen of the differentiation of the future gonad, either histologically or morphologically, in the direction of maleness (to form a testis) or femaleness (to form an ovary). It should also be noted that under normal circumstances, even at this stage of gonadal differentiation, a relatively small number of the primordial germ cells are commonly recognised as they pass along their normal migratory pathway, because they have yet to enter the gonadal region. These may still be located, for example, within the dorsal mesentery of the hindgut or in the medial coelomic bay. It is, however, most unusual to see evidence of primordial germ cells migration elsewhere. If they survive in extra-gonadal sites, they may give rise to teratomas.

At about TS17-18 (10.5-11 dpc), the mesonephros is at its largest size, and clearly contains both mesonephric tubules and an obvious laterally located mesonephric portion of the nephric duct. What is also noted at about this time, is the first obvious evidence of differentiation of the superficial layer of coelomic mesothelium overlying the gonadal primordium. The mesothelium in this location later shows clear evidence of proliferation, and is seen to invade the subjacent mesenchyme tissue. To form what are termed the cortical cords or the primary sex cords. In the male, but not in the female, the primary sex cords proliferate within the medullary component of the future testis, giving rise to the testicular cords, and these later differentiate to form the seminiferous cords (seminiferous tubules). These completely surround the primordial germ cells and their division products, the pre-spermatogonia and spermatogonia. By this time, almost all of the primordial germ cells have migrated into the future gonadal region of the urogenital ridge. The seminiferous cords develop exclusively and characteristically within the future medullary region of the gonad. By contrast, in the female, the impression is gained that the primary sex cords completely regress at this stage, and a second wave of coelomic mesothelium differentiates and proliferates at a slightly later stage of gonadal differentiation, to form the so-called secondary sex cords or germinal epithelium. Consequently, when this tissue invades the subjacent mesenchyme, it then surrounds both the primordial germ cells and their division products, such as the germ cells of the ovary (premeiotic germ cells/ oogonia) and later the meiotic germ cells (oocytes), to form the first evidence of primordial follicles. Characteristically, in the female, these are concentrated in the peripheral part of the future gonad, and this is destined to form its future cortical region. Shortly afterwards, the medullary region of the ovary almost completely regresses. Clearly, at this stage, both the ovary and testis contain mesenchymal stroma.

By about TS20 (12 dpc), the earliest evidence of sexual dimorphism within the gonad is seen, so that by about TS21 (13 dpc) it is now possible to recognize for the first time whether the gonad is destined to develop as a testis or as an ovary. As noted below, although difficult to discern, the presence of Barr bodies in the female germ cells would, in fact, represent the first evidence of sexual dimorphism in the female. Under normal circumstances, however, it is the clear evidence of the testicular cords that are soon destined to form the seminiferous cords that are observed in the testis that gives the future testis a “striped appearance” and this is normally considered to be the first obvious evidence of sexual dimorphism. This is the characteristic feature that is not observed either at this stage, or in future stages, of ovarian differentiation; the ovary at all stages of its development is seen to possess a “spotty” appearance. What is also apparent by about TS20 (12 dpc) is that much of the rostral part the mesonephros has regressed. Mesonephric regression occurs quite rapidly, once it is initiated, and the first evidence of this phenomenon is seen at about TS18-19 (11-11.5 dpc). By about TS21 (13 dpc), the majority of the rostral part of this organ has dramatically decreased in volume, while during this time the gonad substantially increases in volume. This relationship is even more apparent by about TS21-22 (13-14 dpc) when the majority of the rostral part of the mesonephros has completely regressed, and it is only then that the most caudal part of the mesonephros shows maximal evidence of differentiation. By about TS22-23 (14-15 dpc), the caudal part of the mesonephros has almost completely regressed, and has largely been replaced functionally by the metanephros.

From as early as about TS13-15 (8.5-9.5 dpc), the two nephric ducts, often called mesonephric ducts, extend caudally in the direction of the postero-lateral parts of the urogenital sinus. Contact is usually made with these regions of the urogenital sinus by about TS16-17 (10-10.5 dpc), when a diverticulum of the nephric duct, termed the ureteric bud, is formed from close to where the caudal end of the nephric duct makes contact with the urogenital sinus. The latter is destined to form the future bladder and pelvic urethra. In both sexes, the nephric duct stimulates the differentiation of a more laterally located duct termed the paramesonephric (Müllerian) duct. The paramesonephric duct grows in a caudal direction along the nephric duct and reaches the urogenital sinus, at the dorsal pelvic urethra by TS21 (13 dpc). Both the nephric and paramesonephric ducts connect to the pelvic urethra at a thickened region of the epithelium called the urogenital sinus ridge. Once formed, the ureteric bud then grows rostrally towards the lowest pole of the nephrogenic ridge and induces it to form the metanephric mesenchyme. The latter rapidly differentiates, so that by about TS19-20 (11.5-12 dpc) early evidence of differentiation of the ureteric bud to form the tubular or drainage system of the metanephros or definitive kidney is evident, while within the metanephros itself, excretory components are first observed. It is also relevant to note the timing of regression of the mesonephros, because the nephric duct is later taken over to form the “drainage” system of the testis. While the paramesonephric duct is retained in the female, it gives rise to the oviduct, the uterus and its cervical region, and most caudally the upper part of the vagina, during development, and the entire vagina in the adult. During development, the lower part of the vagina, called the sinovaginal bulb, develops from the urogenital sinus ridge. However, the sinovaginal bulb is a transient structure and does not contribute to the adult vagina. In the male, the urogenital sinus ridge becomes the verumontanum and the paramesonephric duct is induced to regress.

As the metanephroi gradually ascend rostrally to their definitive position in the upper and postero-lateral parts of the abdominal cavity, one on either side at the level of the first few lumbar vertebrae, so both the ovaries and the testes gradually migrate towards their definitive location. While the ovaries in the mouse are finally located just postero-lateral to the lower poles of the metanephroi, and by the time of birth are completely encapsulated, so the testes descend caudally. Initially the testes descend into the pelvis, and by term they are usually located infero-laterally, one on either side of the bladder. The associated structures such as the prostate glands are first apparent in the male shortly before birth, at around TS25 (17 dpc), and their development is also discussed in more detail below.

It is evident that a close and complementary relationship clearly exists during the early stages of development in the mouse between the urinary and the reproductive systems. In both sexes, the nephric duct plays a critical role in the differentiation of the paramesonephric duct. In the male, the mesonephric ducts and mesonephric tubules differentiate to form much of the “drainage” system of the testis, such as the rete testis, the efferent ducts, the epididymis and ducti deferens, while the paramesonephric duct is induced to regress. In the female, by contrast, while the paramesonephric ducts give rise to much of the internal genital duct system, it is the nephric ducts that largely regress. Bearing these relationships in mind, it is now appropriate to consider the various components that are noted during gonadal differentiation in both sexes, and only then will it be appropriate to consider the differentiation of the internal genital duct systems and the external genitalia in more detail. Only then will it be possible to provide a summary of these events in relation to the more exact timing of their appearance and differentiation in both sexes of this species. Because many of the terms used in the literature, albeit synonymous with the terms used here, differ considerably from them, it would seem to be reasonable to put these alternative terms in parentheses in association with those selected to be employed in the GUDMAP terminology.

Primordial germ cells

The primordial germ cells are first clearly observed at about TS11 (7-7.5 dpc), during the early post-implantation period, in the inner or mesodermal component of the wall of the secondary yolk sac, being most readily seen if this region is stained histochemically to demonstrate the presence of intracellular alkaline phosphatase enzyme activity. They may also be recognised, albeit with greater difficulty, at this stage of development, by their large ovoid morphology, being somewhat larger in volume than their surrounding cells. Initially about 50 of the precursors of these cells may be recognised at the primitive streak stage, in the wall of the secondary yolk sac. By TS11-12 (7.5-8 dpc), however, the number of primordial germ cells present is nearer to 150, and these cells are now mostly located in the wall of the yolk sac close to the base of the allantois and at the caudal end of the primitive streak.

Some authorities have reported the presence of a Barr body at the periphery of the nucleus of female primordial germ cells at this stage of development, representing the presence within these cells of the inactivated X-chromosome. It is sometimes stated that this structure is more easily seen when these cells are stained with toluidine blue, which also stains their basophilic cytoplasm characteristically. By about TS12 (8 dpc), evidence of migration of the first of these cells is noted, believed to be initially by amoeboid movement, and later by possibly other mechanisms. Their migratory pathway from the base of the allantois to their definitive location in the gonadal region of the urogenital ridges is initially via the endoderm of the hindgut. Their migratory pathway is then via the dorsal mesentery of the hindgut and medial coelomic bay to the gonadal region of the urogenital ridge. The first primordial germ cells reach this region by about TS15 (9.5 dpc), while the majority tend to reach the gonadal region by about TS20-21 (12-13 dpc). These cells increase in number mitotically, so that it has been calculated that by about TS22 (14 dpc) there may be as many as 25,000 of germ cells present in the gonads.

The indifferent gonad stage

As indicated above, the first evidence of the gonadal primordia is seen at about TS18 (11 dpc), and over the next one or two days they become invaded by the primordial germ cells. By about TS19 (11.5 dpc), the mesothelium overlying the gonads differentiates and proliferates to form the cortical cords or primary sex cords that, in the male, give rise to the testicular cords that later are destined to form the seminiferous cords (seminiferous tubules). These develop within the future medullary region of the testis. In the female, the primordial germ cells tend to concentrate in the more peripheral part of the gonad, and the medullary region largely regresses. What is also evident in the female, is that the initial round of proliferation of the gonadal mesothelium (also termed, although incorrectly, “germinal” epithelium) largely regresses by about TS19-20 (11.5-12 dpc), and does not appear to be associated with an invasion of the subjacent mesenchyme tissue at this stage. In testes at about TS21 (13 dpc), but not in the ovaries, there also appears to be an increased degree of vascularity that is particularly evident in their peripheral margin. This is seen very shortly before the most obvious event associated with sexual dimorphism is apparent, with the first evidence of the “striped” appearance of the future medullary region of the testis, due to the presence within this region of the seminiferous cords.

In the ovaries at about TS21 (13 dpc), the germ cells tend to cluster together, and these are uniformly distributed throughout the ovary. At about this stage of ovarian development, evidence of a second wave of cellular proliferation just subjacent to the surface of the gonad is seen. In the testes, at about this time, the testicular cords incorporate both the primordial germ cells and some of the mesenchymal cells. The primordial germ cells undergo intense mitotic activity at this stage, to form the prespermatogonia and, by about TS25-26 (17-18 dpc), these cells are destined to give rise to spermatogonia. The mesenchymal cells that have also been incorporated into the testicular cords are destined to give rise to the pre-Sertoli and later the Sertoli cells or sustentacular cells. Pre-Sertoli cells are SRY/SOX9 positive cells that are not incorporated into testis cords; they differentiate into Sertoli cells in the process of enclosement into the cords and not as stated here”. By about TS21 (13 dpc), the gonads are now readily recognised histologically as being either testes or ovaries. The shape and size of the gonads also differs slightly and this is particularly evident by TS22-23 (14-15 dpc), as the testes tend to be more rounded in shape, while the ovaries tend to be somewhat elongated and ovoid in shape.

The testis and its immediate drainage system

The cortical cords that give rise to the primitive testicular cords will later differentiate into the seminiferous cords (seminiferous tubules). These form septae that will subdivide the testes perpendicularly into discrete longitudinal units. The testicular cords will shortly afterwards become associated with the tubular tissue located in the hilar or mediastinal region of the testis, termed the rete testis, and this will, considerably later, effectively “drain” the sperm that differentiates within the seminiferous cords into the efferent ducts. Drainage of the “morphologically mature” spermatozoa subsequently occurs into the mesonephric duct-derived drainage system of the testis, although such spermatozoa need to undergo exposure to certain substances within the female tract before they are capable of fertilising an egg (see below). At this stage, the interstitial cells are located in the regions between the seminiferous cords.

It is now also generally accepted that the tubular system that constitutes the rete testis is also derived from the mesonephric duct. The Sertoli cells that differentiate from the mesenchymal cells that are also incorporated within the seminiferous cords have a critical role to play during the spermiogenesis phase of spermatocyte development, in that they act as “nurse” cells that facilitate sperm “maturation.” It is believed that the Sertoli cells are stimulated by Follicle Stimulating Hormone (FSH) to release androgen-binding protein that prompts the spermatogenic cells to bind and concentrate testosterone that, in turn, stimulates spermatogenesis. The source of the testosterone and other androgenic hormones needed for this process is derived from the Leydig cells. These cells are differentiated from a specialised proportion of the mesenchymal cells that remain in the interstitial tissues, and all of the interstitial cells are located outside and between the seminiferous cords. An additional role of the testosterone and the other androgenic hormones produced by the Leydig cells is that they play a critical role in stimulating both the internal genital duct system as well as the external genitalia to develop in the direction of maleness. Regression of the paramesonephric ducts (Müllerian ducts) is also induced in the male. This effect is produced by the influence of Müllerian-inhibiting substance (MIS) or anti-Müllerian hormone (AMH).  This hormone-like substance is produced by the precursors of the Sertoli cells of the cortical cords or the primitive testicular cords. The differentiation of the derivatives of the mesonephric ducts is driven by androgens produced by Leydig cells.

By about TS22 (14 dpc), while the mesonephros–derived “drainage” ducts, such as the rete testes, efferent ducts and epididymes, as well as the ducti deferentia, are by now all canalised to some extent, the seminiferous cords (seminiferous tubules) have yet to show evidence of canalisation. These appear to be solid cords of tissue in which the germ cells and the pre-Sertoli cells are embedded. What is also evident at this stage, is that the dorsal mesentery of the testis, formerly the urogenital mesentery, tends to become narrower. After about TS21 (13 dpc), once the testis is recognised, it should then be termed the mesorchium. Similarly, the mesentery that supports the ovary should now be referred to as the mesovarium. Equally, at about this stage, the testes become ovoid in appearance, and are located close to the anterior part of the upper half of the metanephroi, and close to the antero-lateral surface of the adrenals. The location of the metanephroi at this stage, as well as the testes, are still close to their sites of origin. By TS22-23 (14-15 dpc), however, the first evidence of “ascent” of the kidneys is seen, and a gradual but progressive separation between them and the testes becomes increasingly evident. It is believed that the kidneys “ascend” as a result of differential growth of the upper compared to the lower half of the embryo, while the testes tend to be retained in the caudal part of the embryo due to the constraining or “anchoring” effect of the gubernaculum. These bands of fibro-muscular tissue connect the lower poles of the testes with the inner aspect of the scrotum (formerly the sacs within the labial folds in the female and the scrotal folds in the male). It is only during sexual arousal in the post-pubertal mouse that it is believed that the gubernaculum plays a role in “guiding” the testis into the scrotum via the inguinal canal. This is unlike the situation in the human, where the testes “descend” to the level of the external inguinal “ring” shortly before full-term, and the testes have usually descended into the scrotal region by the time of birth.

It is also clear that in the mouse, as in many other mammalian species, when the testes are maintained at core-body temperature for the majority of the time, this does not appear to have a detrimental effect on spermatogenesis. This clearly contrasts with the situation in the human, where it is believed that a temperature of some degrees below the core-body temperature is essential for normal spermatogenesis to occur. Indeed, in the human, when one or both of the testes remain in an intra-abdominal location, there is said to be a significant risk of malignant changes in the germ cell population, to induce the formation of teratocarcinomatous changes.

In the mouse, the first evidence of differentiation of the outer region of the testis to form a fibrous capsule, or tunica albuginea, is observed at about TS22-23 (14-15 dpc). The cellular elements from which this forms are located just subjacent to the outer coelomic epithelial covering of the testis (the future tunica vaginalis testis) is first noted at about TS25-26 (17-18 dpc). Large numbers of spermatogonia, many of which are in division, are also recognised within the seminiferous cords at this stage. Similarly, at about TS26, the first evidence of differentiation of the external genitalia is also seen (see below).

By about TS24 (16 dpc), the testes appear to have “descended,” and are now located one on either side of the upper part of the sides of the bladder. At about this time, the tubular arrangement of the epididymis is also clearly seen, with its distal elements draining into what is now termed the ductus deferens. The mediastinum testis is also first recognised at this stage, in what was formerly termed the hilar region. Another structure that is also usually present by the end of this stage is the appendix testis, one of the derivatives of the paramesonephric duct that is seen in the male. This appears to be in the form of a blind-ended diverticulum, located at the rostral end of the epididymis. The other structure is the prostatic utricle (also termed the uterus masculinus), which is derived from the urogenital sinus (possibly as a male homologue of the sinovaginal bulb). Apart from these two structures, one at least of which is believed to be a derivative of the paramesonephric duct, the rest of the latter has by this stage completely regressed in the male. It should also be noted that an appendix epididymis is also present, and believed to be a mesonephric derivative that is located in close proximity to the appendix testis. No function has yet been assigned to these structures.

By about TS24 (16 dpc), the first stages of differentiation of the urethra are seen. All of the epithelium lining the urethra, in both males and females, is derived from cloacal endoderm. The urogenital sinus which develops from the anterior region of the cloaca gives rise to the bladder and pelvic urethra, whilst the phallic urethra, develops from a bilaminar ventral outgrowth of the cloaca, called the urethral plate epithelium (urethral plate). Later, the phallic urethra will develop into the penile and clitoral urethra, in males and females respectively. In males, the two ejaculatory ducts, that probably represent the most caudal part of the ductus deferens, enter the prostatic urethra, at the verumontanum one on either side of the prostatic utricle.

By about TS25 (17 dpc), the seminal vesicles are first noted and are believed to be derived from rostrally directed diverticulae that develop close to the distal ends of the nephric ducts, just proximal to the ejaculatory ducts. The seminal vesicles subsequently secrete a fructose-rich fluid that, with the testicular fluid constitutes a considerable proportion of the seminal ejaculate, while the prostate gland begins to form at about TS25 (17 dpc) and the fluid it secretes also adds bulk to the ejaculate. By about TS25-26 (17-18 dpc), the first evidence of canalisation of the seminiferous cords is seen. The prostate gland is formed from four principal primordia, called prostate buds, subdivided into anterior, ventral, dorsal and lateral regions. The most dorsal components, which will become the dorsal prostate gland, are also called coagulating glands. The coagulating glands lie in close proximity to the concave smooth surface of the seminal vesicles. The ducts from the coagulating glands open into the prostatic region of the urethra close to the openings of the ejaculatory ducts. It should be noted that only minimal evidence of differentiation is observed in these glands and the other components that give rise to the prostate during the early post-natal period, as the role of the secretions of the former coagulate the ejaculate in the proximal part of the female tract. This forms the so-called “vaginal plug,” a feature characteristically seen in the mouse and in various other (but not in all) rodents. Curiously, this particular feature is less evident in the rat.

Evidence of differentiation of the urethra has also been noted, as well as early evidence of development of the bulbo-urethral glands, and the prostatic utricle. Bulbourethral glands and numerous urethral glands develop in both males and females. Bulbourethral glands are larger and urethral glands more numerous in males, compared to those in the female. At the histological level, some of the germ cells have differentiated to form spermatogonia, while in the interstitial tissue between the seminiferous cords a few Leydig cells may also be recognised.

Development of the ovary and the internal genital duct system in the female

As indicated above, the differentiation of the ovary is less obvious than that of the testis. This is principally because no histological equivalent of "stripes" is observed within it. Once sexual dimorphism has occurred, at about TS21 (13 dpc), the location of the ovaries and testes is similar to that observed previously. Very shortly after this period, however, the kidneys begin to "ascend" to their definitive location, while the ovaries remain in a position close to the site of their first appearance. They appear to be "fixed" in this position, possibly due to the anchoring effect of the ovarian ligament, which probably represents the homologue of the gubernaculum. Little evidence of the round ligament of the uterus is seen at this stage. Unlike the situation in the human, the ovaries in the mouse fail to "descend" into the pelvis. 

At about TS24 (16 dpc), the ovaries are usually located just lateral to the lower half of the kidneys. What is also evident at this stage, is that while their rostral and dorsal surfaces appear to be closely embraced by the flattened tissue of paramesonephric origin, the rest of the ovary appears largely to be exposed. By this stage, the ducts that are destined to form the uterine horns have yet to canalise. It should also be noted that during their course, they pass in front of the ureters. By about TS25 (17 dpc), the upper third or half of the ovary is now enveloped by this flattened tissue. By TS26 (18 dpc), most of the lateral part of the upper two-thirds of the ovary is now enveloped by this tissue, and only by about 18.5 dpc is the ovary seen to be completely surrounded by the flattened tissue of paramesonephric duct origin. This therefore corresponds to the first stage when the ovary is either completely or almost completely surrounded by a thin ovarian bursa (capsule). At this stage, both the ovary, as well as the precursor of the oviduct, are now located within this bursa. At the time of birth, the latter, with its contents, is either partially or completely hidden behind the lower pole of the kidney.

It has also been noted that the first evidence of differentiation of the muscular region of the wall of the uterine tubes is seen at about TS25 (17 dpc) to form the myometrium, while the cervical region of the uterus also shows early evidence of differentiation.

Between TS24 (16 dpc) and TS26 (18 dpc), the overall volume of the ovary appears to remain fairly static. At the earlier period, its overall length is about half of that of the kidney, but by the later period, it is closer to 20% of the length of the kidney. This is principally because the kidneys appear to be substantially increasing in volume over this period. While the ovaries remain ovoid in shape during this period, the most obvious feature observed is therefore the progressively increasing part of their surface that becomes covered by the thin membrane of paramesonephric duct origin. Similarly, the two paramesonephric ducts (the future uterine horns) are directed caudally and towards the midline, and meet just behind the trigone of the bladder, although before birth their two intervening walls separate their lumina in this location. While the upper part of the vagina is derived from the caudal part of the fused paramesonephric ducts the lower part of the developing vagina, called the sinovaginal bulb, is derived from the urogenital sinus and develops from the urogenital sinus ridge. During development, the upper and lower vagina migrate caudally along the pelvic urethra towards the clitoris and because the bulb is a solid epithelial cord, the vaginal opening doesn’t form until after birth. In the adult, the entire length of the vagina develops from the upper vagina and has a paramesonephric duct origin, whilst the sinovaginal bulb contributes to part of the vulval epithelium.

The ovarian follicles are of two types, and both are principally located within the ovarian cortical component of mesenchymal stroma. There are those do not grow, and the majority belong to this class, and those in the second class that show evidence of growth. Of the latter class, it has been calculated that only a very small proportion of these is likely to be ovulated. What is also evident either at this time or shortly after birth is that many of the oogonia within the ovary appear to have entered prophase of the first meiotic division to become meiotic germ cells, although few if any of these are destined to be ovulated. It is also of interest to note that few if any of the nephric duct derivatives are usually observed in the female mouse, as this system normally completely regresses. While the rete ovarii is derived from the nephric duct, this usually completely regresses. Accordingly, the only named derivative of the nephric duct in the female is the epoophoron. This may form connections with the rete ovarii, and may persist throughout life. In the human female, the mesonephric duct may also persist into postnatal and even into adult life as Gartner’s duct, and this may occasionally give rise to cysts. It is unclear whether this also occurs in the mouse. These mesonephric derivatives are located in the broad suspensory ligament (or mesometrium) from which the uterine horns are suspended, and are therefore located along the original line of descent of the nephric duct.

The development of the external genitalia

The early stages in the development of the external genitalia are similar in the two sexes. As noted above, possibly up to about TS24 (16 dpc) it is not yet possible to recognise the sex of the developing embryo from its external appearance, since the both sexes initially appear identical. However, at around TS23 (15 dpct) minor anatomical differences are observed such as the slightly larger size of scrotal swelling and perineum in males and the larger distance between the urethral duct and anus. It should be noted, however, that even at full term, because of the immaturity of the mouse conceptus at that time (compared, for example, to the situation in the human conceptus at full term), this is still not a simple exercise (see below). About TS22-23 (14-15 dpc) in the mouse approximately corresponds to the end of the so-called “embryonic” period in the human conceptus. It should also be recalled that while the eyelids open in the mouse at about two weeks after birth, they open in the human at about 28 weeks of gestation.

A genital tubercle is first apparent at about TS19 (11.5 dpc) while the two ventrolateral genital swellings and the dorsal genital swelling are first noted at about TS17-18 (10.5-11 dpc). The swellings later fuse rostrally in the region of the genital tubercle. Lateral swellings, called preputial swellings develop on the genital tubercle of males and female. These enclose the preputial glands and will grow ventrally around the tubercle and develop into the prepuce, which surrounds both the penis and clitoris in the adult mouse. At the base of the genital tubercle, paired scrotal and labial swellings develop in males and females respectively, later becoming the scrotum and labia.

In humans, in the male, the scrotal folds also fuse across the ventral midline to form the scrotal folds (or scrotal sac), while in the female these do not fuse across the ventral midline. The urethral folds, one located on either side of the urethral groove, in the human form the labia minora, while the labial folds give rise to the labia majora. It should be noted that not even the labia majora are well developed in the mouse. However, external genitalia development in the mouse is different to humans. In mice, a phallic urethra develops in both males and females, from cavitation of the urethral plate epithelium, which progresses in a proximal to distal wave. The phallic urethra is located within the penis in males and clitoris in females. This is in contrast to humans, where mesenchymal growth from paired urethral folds rolls up the urethral plate into a tube, in the process creating a large urethral groove, and only males develop an external urethra.

Unlike the situation in the human, an os penis develops in the mouse after birth, as does the erectile tissue around the penile urethra in this location. The latter forms the corpus cavernosa penis. In females, a much smaller structure, homologous to the os penis, forms in the clitoris. Despite the events associated with the development of the external genitalia in mice, it is still extremely difficult to distinguish between the sexes in the newborn period, although the distance between the anus and genital tubercle (the immature phallus) is usually slightly less in the male than in the female. Such a distance (AGD) has been often used as a parameter of sexual differentiation in teratology.

Observations on reproductive development obtained from the analysis of staged histologically sectioned mouse embryos

As previously, with regard to the development of the renal system, the first stages when the development of specific features of the genital system are observed are noted here. These have principally been obtained from the detailed analysis of serially sectioned mouse embryos isolated at sequential stages of development.

TS10 (about 7 days post coitum (dpc)): The first evidence of the primordial germ cells are seen histologically in the mesodermal component of the wall of the secondary yolk sac towards the end of this stage.

TS11 (about 7.5 dpc): Towards the end of this stage the number of primordial germ cells present in the wall of the secondary yolk sac is in the region of 150. They are by now mostly located close to the allantois, although some are located at the caudal end of the primitive streak. The first evidence of a gonadal primordium is seen at about this stage. They have yet to be invaded by the primordial germ cells..

TS12 (about 8 dpc): The hindgut diverticulum is first recognised at this stage.

TS13 (about 8.5 dpc):[for reference only] The blind-ending primitive foregut makes contact with the surface indentation termed the oral pit (or stomatodaeum) at about this stage, or during the early part of the following stage. A bilayered membrane, termed the buccopharyngeal membrane initially separates the two, and this usually breaks down during TS14.

TS14 (about 9 dpc): The first evidence of differentiation of the pronephros from the intermediate plate mesoderm is seen at this stage, and slightly later the pronephric duct develops. Shortly afterwards, the pronephros regresses, and is replaced during the next stage by the mesonephros. The pronephric duct is then taken over by the mesonephros, and is then termed the nephric duct (or Wolffian duct). The buccopharyngeal membrane breaks down.

TS15 (about 9.5 dpc): Some primordial germ cells have reached the future gonadal region of the urogenital ridge by this stage. However, most of the primordial germ cells are observed at some stage along their migratory pathway. These cells are either in the endoderm of the wall of the hindgut, or in the dorsal mesentery of the hindgut. They may also be seen passing across the region of the medial coelomic bay. Those in the latter location would shortly enter the future gonadal region.

TS16 (about 10 dpc): The genital (or gonadal) ridge is first clearly evident as a longitudinally running ridge or elevation that extends along the majority of the medial aspect of the urogenital ridge at this stage. Vesicles are first recognised in the mesonephros as well as mesonephric tubules, and the former are connected via the tubules to the mesonephric portion of the nephric ducts.

TS17 (about 10.5 dpc): With regard to the development of the future bladder, the first evidence of the urorectal septum is noted, and by about TS19/20 this will separate the cloaca into a dorsal (hindgut) and a ventral (urogenital sinus) component. An endodermal invagination comparable to the oral pit (see TS13) is observed in the region of the cloaca at this stage. The bilayered membrane that separates it from the endodermally-lined cloaca is then termed the cloacal membrane.

TS18 (about 11 dpc): The earliest evidence of the metanephric mesenchyme is seen at this stage, within the most caudal part of the nephric component of the urogenital ridge, and is induced by the ureteric bud. The first evidence of the so-called gonadal primordium is also seen at this stage, and replaces the genital primordium (or gonadal ridge). Towards the end of this stage and during the first part of the following stage, the cloacal region will become divided into two parts by the down growth of the urorectal septum to form the hindgut dorsally, and the urogenital sinus ventrally. Similarly, the cloacal membrane will also become divided into two parts.

TS19 (about 11.5 dpc): The first wave of cortical activity in the surface region of the future testis occurs at this stage to form the cortical cords, and these will in due course subdivide the testes perpendicularly into discrete longitudinal units that are destined to form the testicular cords. Towards the end of this stage, and during the early part of the following stage, the urorectal septum will completely separate the cloaca into dorsal and ventral components. These represent the primitive hindgut, and urogenital sinus, respectively. The metanephric mesenchyme is first clearly evident at this stage, with early evidence of both excretory and drainage components (future collecting duct system), the latter differentiating from the rostral part of the ureteric bud. Towards the end of this stage, the first evidence of the differentiation of the ureteric bud to form the definitive ureter is seen. The first appearance of the genital tubercle is noted at this stage. This will in due course give rise to the penis in the male and the clitoris in the female.

TS20 (about 12 dpc): The majority of the primordial germ cells have reached the future gonadal region of the urogenital ridge. The first evidence of a Barr body may be seen in appropriately stained female germ cells. The increased down growth of the urorectal septum towards the end of this stage now completely separates the cloacal membrane into a dorsally located anal membrane, and a ventrally located urogenital membrane. These two membranes then become separated in the midline by a fibrous structure that is called the perineal body in humans. Much of the rostral part of the mesonephros shows evidence of regression, and regression of the entire mesonephros becomes increasingly apparent during the next few stages.

TS21 (about 13 dpc): The gonads are first histologically recognisable as being either testes or ovaries at this stage. In the testis (male embryos only), the cortical cords have now developed into the testicular cords, and have further differentiated into the seminiferous cords. The germ cells undergo active mitotic division, so that prespermatogonia and occasionally spermatogonia may be recognised. Some of the mesenchymal cells that are located within the seminiferous cords (seminiferous tubules) are now recognised as developing Sertoli cells. In the ovary (female embryos only), the first evidence of rete ovarii may be seen in the hilar region of the ovary at this stage, as well as a distinct paramesonephric (or Müllerian) duct in association with the most lateral part of the mesonephros. Once the gonads are recognised as either testes or ovaries, their gonadal mesenteries are thereafter termed either the mesorchium or mesovarium, respectively. The urogenital sinus (the future bladder) now possesses a distinct vesical part, with an urachus at its apex that is directed towards the umbilical region.

TS22 (about 14 dpc): During this stage, the first evidence of the definitive bladder is seen, (although the region of the trigone cannot yet be recognised). Early evidence of the pelvic and phallic parts of the urogenital sinus is also noted. The earliest evidence of the labial or scrotal folds is seen, and these will give rise to some of the components of the external genitalia in both sexes, although the presence of the genital tubercle has already been noted at TS19. In the male, the ventral part of the scrotal swellings fuse across the midline to form the scrotum, while in the female the equivalent labial swellings remain separate. The labial swellings will give rise to the labia. The caudal part of the mesonephros shows almost complete evidence of regression, and has largely been replaced by the metanephros.

TS23 (about 15 dpc): The first evidence of differentiation of the distal part of the genital tubercle is seen, with the differentiation of the glans penis. The preputial swelling is evident at this stage. It basically reaches to the distal region around E 15.5.The testis appears to be divided into a cortical region and a medullary region. It is within the latter that the primitive seminiferous cords (seminiferous tubules) continue to differentiate. A gubernaculum testis is also first recognised at this stage.

TS24 (about 16 dpc): The trigone region of the definitive bladder is first recognised at this stage. It is at the apex of the caudal part of this region where the mesodermally derived mesonephric ducts insert into the future prostatic region of the urethra, and at this location, they will give rise to the ejaculatory ducts. Both the appendix testis and appendix epididymis is first recognised during the latter part of this stage. The caudal parts of the ureteric buds (the future ureters) insert into the upper and outer part of the trigone region. Within the ovary, some of the germ cells have achieved the oogonial stage of their differentiation, while others may achieve the primary oocyte stage. The oviduct, uterine horn and upper part of the vagina display early evidence of differentiation at this stage.

TS25 (about 17 dpc): It has been calculated that there may be as many as 25,000 germ cells in the gonads at this stage. Unlike the buccopharangeal membrane that breaks down during TS14, the anal membrane usually breaks down during the latter part of this stage. The rostral and dorsal surfaces of the ovary begin to be embraced by the flattened tissue of paramesonephric duct origin. Early differentiation of the cervical region of the uterus is noted at this stage, as well as differentiation of the muscular layer of the uterine horns (the myometrium).

TS26 (about 18 dpc): Early differentiation of the prostate gland has been noted at this stage and, within the bulbar region of the urethra, the bulbo-urethral glands. Within the interstitium of the testis, the occasional fetal Leydig cell may be recognised, while Sertoli cells envelop the seminiferous cords (seminiferous tubules). Early evidence of differentiation may also be seen within the fibrous capsule of the testis, to form the definitive tunica albuginea. In relation to the external genitalia in the male, both penis differentiation and development of the scrotum is also apparent. By the end of TS26, and the period shortly before term, the flattened tissue of paramesonephric duct origin that began to embrace the ovary in TS26 completely surrounds it and forms the thin ovarian bursa that completely surrounds the ovary.