Development of the Gonads

Primordial germ cells originate from the primitive ectoderm and, initially in their development, migrate out of the embryo into the yolk sac. By the 6th week, these primordial germ cells have migrated into the genital ridges of the intermediate mesoderm, multiplying by mitosis as they migrate. The genital ridges are located just ventromedial to the mesonephros. Somatic cells develop from the thickened coelomic epithelium of the genital ridge, and they surround and nourish the primordial germ cells, forming gonadal cords. By the end of the 6th week, male and female gonads cannot be distinguished, and the gonad is sexually indifferent.

In the absence of a Y chromosome and lack of expression of the SRY gene, the mesonephric duct atrophies and the gonadal cords degenerate. The germ cells differentiate into oogonia. The oogonia multiply by mitosis so that there are approximately 7 million by 20 weeks. They spontaneously enter the first phase of meiosis and arrest in prophase where they are called the “primary oocytes.” The granulosa cells develop out of the gonadal cord cells and surround the primary oocytes to form the primordial follicle. Steroidogenic theca cells develop in the surrounding ovarian stroma. There is progressive follicular atresia from the 16th week onward, resulting in approximately 2 million oocytes at birth. Germ cell development is then arrested until puberty.

The gubernaculum is an embryonic structure thought to connect the internal genital system to the inguinal abdominal wall in early fetal development. It is known that the male gubernaculum plays a role in the descent of the testicles into the scrotum; however, the role of the gubernaculum in females is less clear. In the absence of testosterone and anti-Mullerian hormone (AMH), the Mullerian ducts develop, interfering with the connection between the gubernaculum and the mesonephros and gonadal suspensory ligaments. The gubernaculum grows over the Mullerian duct and the muscular fibers become incorporated into the Mullerian duct at the uterotubal junction, forming the origins of the round ligament. The caudal gonadal suspensory ligament becomes the ovarian suspensory ligament with the ovary descending, but only to the level of the pelvis [1].

Development of the Mullerian Ducts and External Genitalia

During the 6th week of development, two paramesonephric (Mullerian) ducts develop just lateral to the mesonephric duct in both male and female embryos. In the absence of testosterone and AMH, the mesonephric (Wollfian) ducts degenerate and the paramesonephric (Mullerian) ducts continue to develop caudally and medially with midline fusion to create the fallopian tubes, uterus, cervix, and upper vagina.

The urogenital sinus forms by week 7. Cells proliferate from the upper portion of the urogenital sinus to form structures called the “sinovaginal bulbs.” These fuse to form the vaginal plate, which extends from the Mullerian ducts to the urogenital sinus. This plate begins to canalize, starting at the hymen and proceeding cranially to the cervix. This process is not complete until 21 weeks of gestation. The hymen remains intact until around 40 weeks, when it spontaneously ruptures to give a patent vagina.

The external genitalia consist of the genital tubercle, urogenital sinus, and the urethral and labioscrotal folds. In females, the genital tubercle becomes the clitoris, the urogenital sinus becomes the urethra and lower vagina, urethral folds develop into the labia minora, and the labioscrotal folds become the labia majora. External genitalia are recognizably female by week 12 of development.

Genetic Control of Sex Development

Sex development can be divided into two processes: sex differentiation, whereby the undifferentiated gonad develops into either a testis or an ovary, and sex determination, whereby the phenotypic sex is determined based on what is produced by the differentiated gonad.

Genes involved in the development of the bipotential gonad have been identified, as knockout mice for these genes have been found to have complete absence of gonads. These include WT1 and LHX9, as well as NR5A1, which codes for steroidogenic factor 1 (SF1) [2].

The differentiation of the common gonadal primordium into an ovary is determined by a complex molecular interplay of pro-ovarian and anti-testis gene expression. In the gonadal ridge of the XX embryo, sexual dimorphism is triggered by R-spondin-1 (encoded by RSPO1) and FOXL2. WNT4, Fst, and β-catenin are also expressed, promoting development of the ovary. R-spondin-1 augments β-catenin signaling, possibly via WNT4 [3].

SF1 encodes a nuclear receptor that plays an important role in the development of the hypothalamic-pituitary-gonadal-adrenal axis. It is thought that SF1 is involved in the upregulation of SRY and SOX9 gene expression, two key testis-promoting genes [4]. DAX1 is a nuclear receptor protein that plays an important role in the development of the ovary. It is encoded by the NROB1 gene located on the short arm of the X chromosome. In mice models, over expression of DAX1 has been found to result in a reduction in the expression of SOX9, possibly via a direct inhibition of SF1-mediated transcription of the gene [5].

Activation of β-catenin signaling prevents SF-1 binding to TESCO in mice, thereby suppressing the male pathway. Additionally, R-spondin-1, FOXL2, WNT4, and β-catenin supress expression of SOX9 and FGF9 (another testis-promoting gene) and therefore prevent differentiation of testes. This antagonistic relationship between SOX9/FGF9 and the ovarian genes is mutual (Figure 2.1) [2,6].

Figure 2.1 Gonad development: Balance of opposing signals [2].

Pathophysiology of Puberty

The onset of pubertal development is heralded by an increase in pulsatile release of GnRH from the hypothalamus. GnRH neurons are known to be mature from birth onward. Following brief activation in the neonatal period, they remain in a dormant state until the onset of puberty. The mechanism for this is thought to be a central inhibition independent of gonadal steroid feedback. The underlying cause of this central inhibition, however, is unclear. Estradiol levels in the stalk-median eminence of female rhesus macaques have been shown to be elevated in the prepubertal state with a subsequential drop in early puberty associated with an increase in GnRH pulsatility, suggesting a possible role for neurestradiol in the central inhibition of GnRH [7].

Pulsatile GnRH release causes gonadotropic cells of the anterior pituitary to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH stimulates production of Androstenedione in the ovarian theca cells, and FSH stimulates the synthesis of estradiol in the granulosa cells under the influence of aromatase enzymes. The pulsatile release of GnRH begins before the onset of pubertal changes. Approximately one year prior to breast budding, prepubertal girls have elevated LH levels during sleep secondary to GnRH pulses. At the start of breast budding, LH and FSH peak amplitudes increase 10-fold and 2-fold, respectively. By Tanner stage 3, basal LH levels are usually detectable during the day. As puberty progresses, these peak amplitudes increase further and estradiol becomes detectable throughout the day. After approximately one year of daily estradiol production, menarche occurs, usually corresponding to the end of Tanner stage 4 [8].

Genetics of Puberty

Kisspeptins, encoded by the KISS1 gene, are a family of neuropeptides that causes activation of the G protein-coupled receptor 54 (GPR54). This receptor is co-localized to GnRH neurons in the arcuate nucleus of the hypothalamus. Kisspeptin signaling via the receptor is thought to play a part in initiating the GnRH pulse generator [9,10].

Neurokinin B (NKB) is a neurotransmitter peptide that is expressed in the same neurons that express kisspeptin. NKB is encoded for by the TAC3 gene, and its receptor NKR3 by TACR3. Mutations in TAC3 and TACR3 have been found to result in hypogonadism [11].

MKNR3 is another gene that has been linked to puberty. A loss of function of the MKNR3 gene has been found in familial cases of precocious puberty, suggesting that MKNR3 plays a role in inhibiting puberty. However, the exact mechanism for this is unknown [12].

It is well known that energy reserves and metabolic conditions play an important role in the timing of pubertal development. Leptin, a hormone released by adipose cells, was first identified in 1994 and is known to play a critical role in body weight homeostasis and the metabolic control of puberty. Patients with mutations in leptin or the leptin receptors have been found to have delayed puberty [13]. The precise mechanism by which leptin influences puberty is not clear, and it may in fact work through an indirect mechanism, possibly through regulation of the KISS1 system [14]. Leptin receptors are highly expressed on kisspeptin neurons. When leptin was administered to leptin-deficient mice, expression of KISS1 increased and puberty was induced [15]. However, this action of leptin on kisspeptin neurons has since been questioned, as LepR knockout mice, in which the leptin receptor was selectively removed from the kisspeptin neurons, still underwent normal puberty [16].

Pubertal Development

Normal pubertal development in girls follows an ordered sequence of events as described by Tanner [17]. Breast development is usually the first sign of puberty. Pubic and axillary hair normally develops about 6 months later, although in a third of girls pubic hair may appear before breast development. Breast development and pubic hair development occur independently, which is a reflection of their underlying driving mechanism. Breast development occurs as a result of rising estradiol levels and pubic and axillary hair by androgen secretion (driven primarily by adrenal glands). Menarche occurs late in puberty, normally corresponding to the end of the growth spurt, when the growth velocity falls below 4cm/year. Maximal growth velocity usually occurs after the start of breast and pubic and axillary hair development, but in some girls the growth spurt may be the first sign of puberty. The complete process of puberty is usually a slow progression, taking a minimum of 18 months [17].

During puberty the ovaries undergo a rapid enlargement as a result of the development of multifollicular cysts under the influence of pulsatile gonadotrophin secretion. The uterus steadily grows through puberty with the body of the uterus gradually becoming longer than the cervix resulting in an adult configuration.

As a result of the relative immaturity of the hypothalamic-pituitary-ovarian axis in the first 2 years following menarche, more than half of menstrual cycles are anovulatory. This results in irregular cycles with cycle frequency varying from less than 20 days to more than 90 days. After the first 1–2 years, the capacity for estrogen-positive feedback on the anterior pituitary develops with the subsequent mid-cycle LH surge and ovulation, resulting in regulation of the menstrual cycle.

Anovulatory cycles are often heavy and prolonged with some girls bleeding for several weeks at a time. This can lead to iron-deficiency anemia, and in rare cases cardiovascular collapse requiring hospital admission and blood transfusion. Initial anovulatory cycles tend to be pain free, although heavy menstrual loss can result in an element of dysmenorrhea. When regular ovulatory cycles commence, the periods often become more painful because of the increased levels of circulating prostaglandins.