Development of Gonads and Germ Cells1

Development of female internal and external genitalia (Flow chart 1.1) occurs independently of gonadal hormones and in absence of testis. Under the influence of SRY gene which is expressed in mesenchymal cells, there is male differentiation despite an internal environment which is rich in estrogen and progesterone.

The mesenchymal cells differentiate into Sertoli cells, and are the first site of expression of SRY gene and several downstream sex determining genes like SOX9 and WTI (Flow chart 1.2). Thus, structurally and functionally intact and endocrinologically competent Sertoli cells are required for optimal SRY expression and testicular differentiation. Low SRY expression due to low Sertoli cell number may result in intersex conditions with ambiguous genitalia like cryptorchidism and hypospadias. Thus, sexual differentiation is actually the differentiation of Sertoli cells. Following this, the steel gene is expressed in Sertoli cells, which help in migration of germ cells by amoeboid movement from wall of yolk sac to the genital ridge by 6th week of development.

Thus, SRY gene codes for transcription factors that regulate expression of downstream autosomal genes (like steroidogenic factor SF1 and SOX9), which regulate Sertoli cell development and differentiation. Sertoli cells induce differentiation of Leydig cells, and secrete Mullerian inhibiting substance (MIS) or Anti-Mullerian hormone (AMH) which causes repression of Mullerian or paramesonephric duct differentiation. The action of hormones secreted by Leydig cells, i.e. testosterone and dihydroxy testosterone (DHT) induces male sexual differentiation of somatic structure, and development and differentiation of external and internal genitalia.

It was believed that sexual differentiation in females is by default mechanism that occurs in absence of Y chromosome.

When primordial germ cells fail to migrate, the gonads remain indifferent or are absent. But in case of testis, it is found that testis may develop even in the absence of primordial germ cells. This is due to the fact that SRY gene on Yp only regulates testicular differentiation, but growth, development and differentiation of male germ cells (spermatogenesis) and spermiogenesis is under the control of azoospermia factor (AZF) on long arm of Y chromosome (Fig. 1.1). This loci consists of 3 subloci AZF a, b and c which regulate specific stages of spermatogenesis and deletion of each loci results in partial or complete spermatogenic arrest.^4–6 So, XX SRY + cases show testicular differentiation due to the presence of SRY, but are azoospermic due to absence of AZF.

There is a striking difference between sexes in timing of gonadal differentiation. Testis organization begins at 6–7 weeks, but ovaries emerge from indifferent stage only at 12 weeks when the earliest sign, the beginning of meiosis appears as evidenced by maturation of oogonia into oocytes.

Paracrine control is the second type of regulation in sexual differentiation. This involves the local dissemination of a hormone or a chemical messenger from its site of synthesis to target cells. There are several chemical messengers which act in the paracrine mechanism. For example in males, meiosis inhibiting substance secreted by Sertoli cells act on surrounding germ cells and arrests their proliferation and differentiation till puberty.

Attainment of full gonadal function requires an intact hypothalamo-pituitary-gonadal axis. This axis functions in the fetus, but is suppressed to very low levels of activity during childhood and gets reactivated during puberty.

Harris GW⁸ in 1964 suggested that hypothalamus or higher neural centers function differently in two sexes. Studies on rat showed that once the male pattern is imprinted on sex centers in the hypothalamus under the action of androgens, the potential for cyclical activity of FSH and LH release by pituitary is lost. However, this testosterone induced differentiation of gonadotropins regulatory mechanism is not seen in humans.

Ovaries are oval (3 × 2 cm) almond shaped structures which lie in ovarian fossa against the lateral pelvic wall. The ovary has a cortex, which in turn, contains germ cells, and a medulla consisting of stroma and blood vessels. In the cortex the ova grow in a relatively hypoxic environment, which is favorable for developing germ cells and prevents free radical induced oxidative damage to germ cells. The stroma consists of connective tissue cells, contractile cells and androgen secreting interstitial cells. There are four types of interstitial cells, primary, secondary, thecal and hilar. These cells have features of steroid secreting cells. These hilar cells contain crystalloids of Reinke and morphologically resemble interstitial cells of Leydig and like Leydig cells secrete testosterone.

After arrival of primordial germ cells (PGC) in the bipotential gonad of a genetic female, there is differentiation of the gonad into ovary. Early in development, the ovary is close to mesonephros which influences gonadal differentiation and is essential for ovarian development. The ovary is not distinguishable till 10th–11th week though fetal testis can be identified by 7th week and PGC forms the oogonia. These oogonia divide rapidly by mitotic division and reach maximum number of 5–7 million by 5th month of gestation. However due to atresia, the number depletes to 1 million till birth and only 400,000 remain at menarche. In women with 45,XO chromosomal complement or 46, XX (Xq deletion), there is accelerated germ cell atresia due to haploinsufficiency of genes on long arm of X-chromosome. The long arm of X chromosome contains two genes (POF 1 and POF 2) which regulate ova growth and differentiation, and are critical for normal ovarian function. Thus two structurally and functionally intact X chromosomes are required for normal ovarian function and optimal oogenesis.⁹ Deletion of long arm of X chromosome 46, XXq del (Fig. 1.2A); 46,XX Iso X [monosomy Xp and trisomy Xq (Fig. 1.2B)] or X monosomy 45,XO (Fig. 1.2C) results in premature ovarian failure and manifests as primary and secondary amenorrhea. In women with 45,XO chromosomal complement, there are streak gonads at birth. In females the mesonephros regresses slowly and ovarian-mesonephros dissociation persists during early ovarian differentiation and Byskov in 1986¹⁰ and Byskov and associates in 1985¹¹ reported that the ovary is invaded by mesonephric cells in the medullary region and they force the germ cells to periphery/ovarian cortex. The advantage of germ cell differentiation occurring in ovarian cortical region is that the developing germ cells are exposed to low oxygen tension and thus are not exposed to high reactive oxygen species (ROS) levels during development. This is in contrast to male germ cells which exist in a state of oxygen paradox and are exposed to high ROS levels due to aerobic metabolism,^12,13 and thus tend to accumulate mitochondrial nucleotide alterations. In a recent study, the authors have shown mitochondrial dysfunction and oxidative stress in cases with premature ovarian insufficiency which leads to accelerated atresia of the germ cells and manifests as premature ovarian failure.

The oogonia divide rapidly until they differentiate to primary oocytes beginning at 8th–10th week of gestation. The primary oocytes begin meiosis, but are arrested at diplotene stage of meiosis I due to meiosis preventing substance or oocyte maturation inhibitor which is secreted by granulosa cells surrounding the oocyte. It is only at puberty that meiosis is induced by 2kD molecule known as meiosis inducing substance. A basement membrane separates the primordial follicle from the stromal cells.

So by 3rd month, the primary oocyte is arrested in diplotene stage of meiosis I and is surrounded by flat epithelial cells known as follicular cells. These flat follicular cells are separated from surrounding stroma by a basement membrane. The oocytes and the surrounding cells do not have direct blood supply and thus the basement membrane surrounding the follicular cells serves a vital function of transfer of nutrients from surrounding microenvironment. At puberty, there are a number of growing follicles that are maintained by the primordial follicles. Each month after menarche, about 15–20 primodial follicles begin to mature and differentiate into primary, secondary, tertiary and finally into mature ovarian follicles.7

In tertiary follicles, there is appearance of fluid filled spaces among the granulosa cells which constitutes the antrum. The antral fluid consists of plasma transudate and secretory products of granulosa cells. Estrogens are found in a much higher concentration in antral fluid than in blood. The granulosa cells and cells of theca interna develop specialized contacts “gap junctions,” which allow small molecules to pass directly between interna and granulosa cells. This is because capillaries do not penetrate the basement membrane surrounding the granulosa cells and oocytes and granulosa cells remain avascular. These gap junctions also allow for synchronized and coordinated functioning of follicular cells.

Under the influence of gonadotropins, the follicle grows in size to form a mature follicle or a graafian follicle. The antral fluid increases and fluid filled spaces coalesce to form a single large antral cavity and the oocyte surrounded by granulosa cells (cumulus oophorus) occupies a polar and eccentric position within the follicle. The average time for development of a primary follicle to antral follicle is 12–14 days. These 15–20 follicles develop into a mature follicle, which is destined either to ovulate or degenerate. In each ovarian cycle, only one mature follicle undergoes ovulation.8

Just 3 hours prior to ovulation, the ova completes first meiotic division and one cell receives most of the cytoplasm and the first polar body receives practically none and forms a secondary oocyte. This secondary oocyte begins meiosis II, but arrests at metaphase stage and the process is only completed at fertilization. If fertilization does not occur, the cell degenerates in 20–24 hours.

The zona pellucida surrounding the oocytes is an acellular glycoprotein membrane secreted by both ova and granulosa cells. It mainly comprises zona protein (ZP); ZP1, ZP2 and ZP3 class of glycoproteins. The zona has species specific receptor sites, which allows only sperm of same species to enter the egg. This feature is very important in animals with external fertilization, but not of much significance in humans.

Following penetration of a single sperm, the zona undergoes certain changes which prevent polyspermy. The zona acts like a filter to allow certain secretory products and uterine milk or embryotroph to enter the zygote, morula and early blastocyst. It also prevents the premature implantation of blastocyst at an ectopic site like fallopian tube.

The zona pellucida subserves a very important function as it keeps the rapidly dividing/cleaving blastomeres together, and maximizes their contact and aids in conception. Compaction among blastomeres is also maintained by zonula occludens and E-cadherins. Theca interna is the inner highly vascularized layer of cuboidal secretory cells. These cells possess feature of steroid producing cells. Cells of theca interna possess LH receptors, and on response to LH they synthesize and secrete androgens. This layer has a lot of blood vessels as is typical of endocrine organs.

Theca interna contains collagen fibers and smooth muscles, and is the outer layer of connective tissue. There is a distinct boundary between theca interna and granulosa layer. This boundary forms a partition between the vascular theca interna and the avascular granulosa layer during the duration of follicular growth.

Several factors are required for oocyte and follicular growth like epidermal growth factor (EGF), insulin like growth factor 1 (ILGF 1), calcium ions and FSH.

Ovulation results in release of secondary oocyte from graafian follicle. This occurs on 14th day of the 28 day cycle. The factors which lead to ovulation are:

Depending on when ovulation occurs, the primary oocyte in primordial follicle is arrested at diplotene stage of meiotic prophase for 12–50 years. During this long period the primary oocyte is exposed to several environmental factors/pollutants that can contribute to meiotic errors and lead to nondisjunction and anaphase lag, and may lead to numerical or structural anomalies like trisomy 21 (Down syndrome).

Spermatogenesis is divided into three phases:

These three phases of germ cell development occur within the seminiferous tubules of the testis. The seminiferous epithelium of germ cells and a population of nonproliferating supporting sustentacular cells called Sertoli cells. Sertoli cell is a polarized cell and gives structural organization to seminiferous tubules and extends full thickness of seminiferous epithelium. Outside the seminiferous tubules, there is connective tissue stroma which contains interstitial cells of Leydig.

The principal piece is 38–40 μm in length and contains fibrous sheath external to coarse fibers and axonemal complex. The end piece comprises of only the axonemal complex.

The sperm released following spermiogenesis are immobile and only gain maturity in the epididymis.

Though sperm can survive several weeks in the epididymis, they survive only for 2–3 days in female reproductive tract. With shedding of majority of cytoplasm during spermiogenesis, sperms lose majority of their antioxidant enzymes and therefore are prone to oxidative injury. However, this is prevented by several enzymatic and nonenzymatic antioxidants in the seminal plasma. Venkatesh et al.¹⁴ in 2009 reported higher ROS levels and low total antioxidant capacity in semen of infertile men.¹⁵ Agarwal et al.¹³ in 2009 reported that infertile men with normal semen parameters have increased ROS levels. This is particularly important when semen samples are processed, washed and prepared for assisted reproductive technology (ART) or intracytoplasmic sperm injection (ICSI). In these cases in the washed semen samples, sperms are exposed to very high ROS levels as the antioxidant rich seminal plasma is removed.¹² Thus it is recommended that ART culture media should be supplemented with antioxidants, though the dosage needs to be regulated so that normal physiological functions like acrosome reaction and capacitation are not impaired. Authors also found after density centrifugation, which segregates spermatozoa with normal morphology from sperms with abnormal morphology that sperm with abnormal morphology produce high levels of free radicals which are detrimental to sperm with normal morphology and thus there is a need to segregate the two population of germ cells after sperm wash in ART to prevent oxidative induced DNA damage in germ cells. They also found that the sperm with normal morphology and motility in semen of normozoospermic infertile men harboured significantly higher DNA damage [DNA fragmentation index (DFI) 23.8%] as compared to sperm of controls (19%). The authors have recently established cut off values of DFI in infertile men at 30% and in male partner of couples with idiopathic recurrent pregnancy loss at 24%.¹⁶10

In the spermatogonial phase, the spermatogonia undergo repeated mitosis and produce spermatogonial progeny. Human spermatogonia based on the appearance of nuclei can be grouped into three types:

For synchronous development of germ cells, all progeny of pale type A spermatogonia are connected by cytoplasmic bridges. For proliferation and cytodifferentiation, male germ require high testosterone concentration and the concentration in the testis is about 200 times than that in the blood.

After several cell divisions, the pale type A spermatogonia differentiate into type B spermatogonia. This is the last event in the spermatogonial phase in the basal compartment of seminiferous tubule.

Meiosis I results in decreasing the chromosomal number to half (23) and meiosis II results in decreasing the DNA content to half.

During meiosis I, there is pairing of homologous chromosomes and formation of synaptonemal complex. This process ensures genetic diversity, as triads which have been modified by crossing over become dyads again. This is the step which distinguishes it from mitosis in which paired chromatids (one old and one newly synthesized) template separate. The movement of maternal and paternal homologous chromosomes to either pole is random and is another source of genetic diversity.

The primary spermatocytes undergo reductional division and form secondary spermatocytes, and shift to the adluminal compartment of the seminiferous tubules. The secondary spermatocyte immediately undergoes 11second meiotic division without entering S phase (synthesizing new DNA). At this stage, though the chromosome number is half there is diploid content of DNA. Thus secondary spermatocyte undergoes equational division to give rise to spermatids with haploid (1n) chromosome number and with single chromatid.

The posterior end of nucleus shows a groove which forms connecting piece/neck region. The centrioles migrate near posterior surface of nucleus. The coarse fibers develop from centriole to attach to nucleus and extend as outer dense fibers peripheral to the microtubules. These fibers unite nucleus with flagellum and thus get the name connecting piece.

The mt migrate from cytoplasm of head region and form a tight helical sheath around the coarse fibers. This is the middle piece. The mt are multitasking organelles and are site of ATP production. They also produce free radicals as by-products during ATP synthesis. As the electron transport chains are localized in the inner mitochondrial membrane, this is the site where ROS are produced and mitochondrial DNA in vicinity of inner mitochondrial membrane is the first site of ROS induced mitochondrial DNA damage. Thus during the course of evolution majority of genes on mitochondrial DNA have translocated to the nuclear genome and only those genes concerned with oxidative phosphorylation are located on the mitochondrial genome. The mitochondrial genome is not protected by histones, and has a very basic proofreading mechanism and a higher turnover rate, thus it tends to accumulate mutations at a much higher rate than the nuclear genome.^15,16 Thus sperm mitochondrial DNA are reduced to disposable elements and are degraded post fertilization. In contrast, the ova develop in a hypoxic environment, and have a quiet metabolism and are not exposed to high ROS levels.

Distal to the middle piece, there are two longitudinal columns of numerous connecting ribs, which surround nine longitudinal fibers of the principal piece and extend till the end of flagellum. A short segment of tail distal to fibrous sheath is the end piece.

During this phase, there is condensation of sperm nucleus, removal of majority of histones (85%) and replacement of transition proteins by protamines (PRM). However, 15% of the genome is bound to histones and 4% of haploid DNA retains its nucleosomal structure. This histone bound portion is located in the periphery of the nucleus, and is less compact and transcriptionally active. It codes for various important developmental genes, which are required during gastrulation like HOX genes and several imprinted gene clusters. However, as this portion is peripherally located, and less compact and condensed it is more prone to oxidative injury.

The size of the nucleus is one-sixth to one-twentieth the size of somatic cell nucleus due to binding by PRM, which neutralizes the charge on DNA and packs it into a compact toroid. At this stage the cell is transcriptionally inactive due to condensation of nuclear chromatin and increased number of sulfide bonds between PRM. Thus, the sperm nucleus is highly compact, condensed and organized, and this protects it from various toxic physical, chemical and thermal insults. Though sperm is believed to be transcriptionally inert, but the peripheral histone bound fraction retains its nucleosomal structure and maintains imprints. Sperm also has certain RNA transcripts, which maintain nucleosomal structure and prevent protamination in specific regions of chromatin. Thus it is believed that RNA may facilitate structural maintenance of open chromatin structure and directly influences eventual gene activation. Also because of shedding of majority of cytoplasm and condensation of 12nucleus, the sperm is very cryostable and can be cryopreserved. However, it has been reported that there is a 30–40% loss of motility in cryopreserved spermatozoa. This is due to depolymerization of microtubules in the sperm axoneme. Microtubules exist in a state of dynamic instability constantly being polymerized and depolymerized, at low temperatures the equilibrium shifts to depolymerized form. On thawing sperm microtubules repolymerize, but are disorganized and this may explain for loss of motility following cryopreservation.¹⁷

In a study done in the author's laboratory, they have found that sperm from infertile men harbor mitochondrial nucleotide alterations. This was seen after whole genome amplification of mitochondrial DNA. These changes were maximal in cases with oligoasthenoteratospermia as compared to cases with just low sperm count. The presence of mutated mitochondrial DNA beyond a threshold count explains for low sperm count due to hypospermatogenesis, and production of sperms with abnormal morphology and impaired motility.¹² However, such infertile men harbouring mitochondrial DNA mutations carry a good prognosis on ART as paternal mitochondrial DNA are not transmitted to offspring; however, such mutated mitochondrial DNA produces increased levels of ROS, which can cause pronuclear block or may impair cleavage.¹⁸

Mitochondria with mutated mitochondrial DNA produce lower ATP levels, but higher ROS levels. High ROS levels induce oxidative stress and damage both mitochondrial and nuclear genome.

In the epididymis, the sperm acquires motility and the ability to fertilize the egg under the influence of androgens. The head of sperm is modified by addition of glycol conjugates, the surface associated decapacitation factor (SDF). This process is known as decapacitation and inhibits the fertilizing ability in a reversible manner.

Once a sperm binds with oocyte plasma membrane there are rapid and slow changes in oocyte which prevent polyspermy. These include depolarization of the oolemma, which results in electrical block to prevent multiple sperm entry. There occur changes in the oolemma polarity due to release of calcium (cortical reaction). The contents of cortical granules are released into perivitelline space.

The cortical granules also release proteases that cleave and degrade oolemma receptors, but also focus perivitelline barrier by cross-linking proteins on the surface of zona pellucida. This is known as zona reaction.

During cryopreservation, there is hardening of the zona pellucida and thus it is difficult to use such ova for ART. But this can be overcome by use of ICSI. Cryopreservation 13also can result in alteration of shape and fracture of zona pellucida and thus can result in polyspermy.¹⁷

Successful fertilization depends upon inherent qualities of the oocyte. However with the advent of ART/ICSI, role of sperm factors in fertilization and embryogenesis is being realized.^19–21 It has been reported that 40% of failed fertilization after ICSI are due to failure of egg to activate. Failed fertilization may be due to centrosomal dysfunction, which leads to disorderly organization of microtubules and to arrest of fertilization. It is also important to realize the importance of structural integrity of intact fertilizing sperm and its contribution to normal human embryogenesis. A high rate of mosaicism was observed is embryos with disrupted sperm.

The zona pellucida acts like a selective barrier and permits fluids from uterine tube and uterine milk/embryotroph secreted from uterine glands to enter the morula. As fluids enter the zona pellucida, it forms a cavity known as blastocoel and is now known as blastocyst. With the development of blastocoel, the blastomeres are segregated into an inner cell mass and a thin outer layer known as trophoblast. At this stage, the cells of inner cell mass are multipotent. These multipotent cells of inner cell mass can form any embryonic tissue but do not give rise to trophoblastic tissue or placenta.

As the blastocyst grows, there is thinning and stretching of zona pellucida and ultimately the blastocyst hatches out of the zona pellucida. Zona proteins prevent premature implantation into the uterine tube of this sticky ball of cells. With its removal, once the blastocyst reaches uterus, it rapidly grows in size and attaches to the uterine endometrium usually at its embryonic pole. This occurs 6 days after fertilization (Day 20–28 of cycle) and marks the beginning of implantation.