## GAMETOGENESIS

### GAMETOGENESIS

1.            GAMETOGENESIS

It is simply the process of gamete production. It is a biological process in which mature haploid gametes are formed from diploid or haploid precursor cells after cell division and differentiation. In gametogenesis meiotic division of diploid germ cells leads to the formation of various gametes, or it may be produced by the mitotic division of haploid germ cells.

In animals

The process of gametogenesis occurs in the specific organs named as gonads which are testis in males and ovary in females. Both male and female individual produces gametes i.e., sperms and ova respectively to achieve the success of sexual reproduction.

The gametes are produced from germ cells that are initially originated in a developing embryo and remain distinct from the rest of the somatic cells. These germ cells are proliferating in gonads via mitosis and lead to gamete formation upon meiotic division.

Male $\rightarrow$ Spermatogenesis $\rightarrow$ Spermatogonia

Female $\rightarrow$ Oogenesis $\rightarrow$ Oogonia.

The variable forms of gametogenesis are because of different sex type.

However, the embryonic development of gametes is same in both male and females before they turn into gametogonia. These gametogonia are the successor of primordial germ cells (PGCs) and are originated from endoderm, while the gonads are mesodermally originated.

1.1.         Spermatogenesis

Spermatogenesis occurs in the seminiferous epithelium. Seminiferous epithelium consists of Sertoli cells (somatic cells) and several types of germ cells.

The process of spermatogenesis occurs in the seminiferous tubules of the testis. The testis is situated in a pouch like a scrotum, to hold them outside the main body. Because spermatogenesis requires 2-3°C less temperature than normal body temperature.

The cytogenesis concerned with spermatogenesis is known as spermatogenesis.

Completion of spermatogenesis

It is divided into two phases

Spermatocytogenesis: In seminiferous tubules the stem cells adjacent to the inner tubule wall divide in a centripetal direction, that means it begins from the outer side and proceeding towards the central region, called a lumen. In humans, testes produce approximately 200-300 millions sperms daily out of which only 100 million become viable and the complete spermatogenesis takes place in 74 days. Spermatogenesis process starts in seminiferous tubules. The seminiferous tubules have two types of cell.

(1)          Sertoli cell

(2)          Germinal cell (Spermatogonia)

The arrangement of  Sertoli cells in the seminiferous tubules divide the seminiferous tubules into two compartments.

(2)          Basal compartment

Spermatogenesis process in the germ stem cells called type A spermatogonia. Type A spermatogonia lie in the basal compartment of seminiferous tubules.

Later on cytogenesis, these cells move in a spiral manner towards lunch of the tubules.

The initial spermatocytogenesis shows divisions where the nucleus is divided, but the cytoplasm of daughter cells is connected via thin cytoplasmic bridges.

The subsequent Type B spermatogonia again divide mitotically to produce diploid primary (1*) spermatocyte. The primary spermatocyte divides meiotically to give else to haploid secondary (2*) spermatocyte. The meiosis II occurs in 2° spermatocyte and leads to the generation of spermatids.

Spermatogenesis: Spermiogenesis is the transformation of a spherical spermatid to a spermlike mature spermatid. It is simply a transformation process, in which the spermatids are differentiated into male germ cells.

Nuclear condensation: The protamine protein is expressed in a high amount in a spermatid. Protamine is rich in arginine and has over 60% arginine residue.

During nuclear condensation, the histones are replaced by protamine. Protamines binds more tightly with DNA and make DNA more condense. This leads to shut down of transcription of all genes. The nuclear condensation resulting in very few transcriptions of the gene, that is approximately 15% of total genes.

Acrosome formation: The vesicles of the Golgi apparatus are merged and invert to form a cap in the most part of the condensed nucleus. That is known as the acrosome.  It functions as lysosome (containing enzymes like hyaluronidase and proteases.

Flagellum formation: One of the centrioles of the cell elongates to become the tail of the sperm.

Sperm cells contain a pair of centrioles. One of the centrioles grows out with nine double peripheral microtubules and two singlets in center. At the time of development of acrosomal vesicle, this flagellum primordium lies opposite to it. It has principally three parts i.e., neck, midpiece and tail. The midpiece is wrapped by the mitochondrial group and the tail with 9 + 2 axoneme.

Cytoplasm reduction: The unnecessary organelles and cytoplasm of developing spermatids is phagocytized by Sertoli cells and disposed of in the tubular lumens. Only a small amount of cytoplasm resides in sperm at neck midpiece region.

Formation of Tail

One of the centrioles of the cell elongates to become the tail of the sperm. A temporary structure called the "manchette" assists in this elongation. During this phase, the developing spermatozoa orient themselves so that their tails point towards the center of the lumen, away from the epithelium.

Spermiation is breaking the structures and bonds anchoring a mature spermatid to a Sertoli cell, so the spermatozoon is released into the tubule lumen and can be washed out of the seminiferous tubule.

Duration

For humans, the entire process of spermatogenesis takes 74 days within including the transport on the ductal system, it takes 3 months. Testes produce 200 to 300 million sperms daily.

Sertoli cell

A Sertoli cell or sustentacular cell is a 'nurse' cell of the testes that provide nutrition to developing sperms. FSH receptor is present on the Sertoli cell. FSH is released by the pituitary gland and stimulates the proliferation of the Sertoli cell. This is the main factor behind the enlargement of testis during puberty. Sertoli cells also act as phagocytes, consuming the residual cytoplasm during spermatogenesis.

Sertoli cells secrete the following substances:

Anti-Müllerian hormone (AMH):–Secreted during the early stages of embryonic development. AMH regress the Mullerian duct in the male.

Inhibin and activins:–Secreted after puberty, and work together to regulate FSH secretion. The inhibin inhibits the secretion of FSH and the activins as the name suggest activates the secretion of FSH.

Androgen binding protein (Testosterone binding globulin)–This protein binds with the testosterone and increases testosterone concentration in the vicinity of seminiferous tubules. The increased concentration stimulate spermatogenesis.

Estradiol:–Aromatase from Sertoli cells convert testosterone to 17 beta estradiol to direct spermatogenesis.

Glial cell line-derived neurotrophic factor (GDNF):–The self-renewal capability is spermatogonia is ensure by GDNF.

The Ets related molecule (ERM transcription factor) – This maintains the stock of the spermatogonial stem cell in the adult testis blood-testis barrier.

7.            Sertoli cell forms the tight junction with each other to form the blood-testis barrier. This protects spermatids from the immune system of the male.

The intercellular adhesion molecules ICAM-1 and soluble ICAM-1 have antagonistic effects on the tight junctions forming the blood-testis barrier. ICAM-2 molecules regulate spermatid adhesion on the apical side of the barrier (towards the lumen). Due to the tight junction, the lumen of the Sertoli cell is differentiated into two compartment

Adluminal compartment this is the lumen where mature sperm are found in basal compartment this is that lumen where spermatogonia cell is found. Tight junction makes an adluminal compartment an immune-privileged site.

Sertoli cells are required for male sexual development. During male development, the gene SRY present on Y chromosome activates SOX9. SOX 9 activates fibroblast growth facter9 (FGF9). The proliferation and differentiation of a Sertoli cell are mainly activated by FGF9. The absence of FGF9 tends to cause a female to develop. Once fully differentiated, the Sertoli cell is unable to proliferate. Therefore, once spermatogenesis has initiated, no more Sertoli cells are created.

1.1.1.     Hormonal Regulation of spermatogenesis –

Leydig’s Interstitial Cells

Leydig cells lie between the seminiferous tubules and act as endocrine cells which produce testosterone in the presence of luteinizing hormone (LH). Testosterone is released into the blood. Leydig cells release a class of hormones called androgens (19-carbon steroids). They secrete testosterone, androstenedione and dehydroepiandrosterone (DHEA) when stimulated by the pituitary hormone luteinizing hormone (LH). LH increases cholesterol desmolase activity (an enzyme associated with the conversion of cholesterol to pregnenolone), leading to testosterone synthesis and secretion by Leydig cells. Testosterone & LH initially act during embryonic development and later at puberty age after LH secretion from anterior hypophysis (pituitary).

The testosterone secreted by these cells along with adrenal cortex initiates the maturation of sperm at puberty.

1.2.         Oogenesis

The formation of ova or egg (female gamete) is known as oogenesis. It occurs in the ovary. Development of ovary specifically governs by fox gene. In female Primordial Germ cell (PGC’s) give rise to egg or ova.

1.2.1.     Oocytogenesis:

Primordial Germ cells (PGCs) arise from posterior proximal epiblast (junction of extraembryonic ectoderm, allantois primitive streak and epiblast) through the BMP signals supply by the extraembryonic ectoderm. Wnts signalling of visceral endoderm make posterior proximal epiblast cells to become responsive (competence) to BMP signalling. Blimpl and Prdml4 genes expression get induced by BMP and both of the gene products require for survival and migration of PGCs up to the genital ridge.

Direct migration of PGCs from epiblast to hindgut (endoderm) takes place during 8th day of fertilization. Tiar protein, an RNA binding protein prevents the apoptosis of PGCs and also helps in the migration of PGCs. Migration is done with the help of fibronectin and integrin protein. Stem cell factor (SCF), c-kit protein and its ligand (KL) are essential for PGC survival and motility.

In female embryo, the gonadal ridge is the site of PGC localization, where they maintain their number through proliferation and get covered by the coelomic epithelium. Proliferating PGC when get established in the developing ovary begin to differentiate into oogonia. Oogonia are responsible for the formation of ova. In female embryo during the fetal development, primordial germ cells (PGC’s) divides mitotically to form a large number of oogonia, between 4th to 8th week of the pregnancy oogonia start to develop and reach up to 7,00,000 (7 million) by the end of 5th month.  Up to 1-2 millions oogonia get declines after 7th month or shortly after birth or at the time of birth.

Thus only 5 million oogonia left which stop dividing and now called primordial follicle and that primordial follicle get differentiated to form primary (1°) oocyte and the primary (1°) oocyte enter in a first meiotic division.

Oogonia

Oocytes form by oogonia, which is stem cells by nature. Oogonia which contain follicle is called primordial follicle. Primordial follicle gets differentiate into primary follicle or primary oocyte undergo meiosis I. Thus completion of oocytogenesis takes place before or shortly after birth as soon as primary oocyte forms. After puberty primary oocytes show growth by receiving the FSH.

Ovary

In mammals, female gonad is called an ovary. Ovary also acts as endocrine gland release steroids, which is responsible for female secondary sexual characteristics and support the pregnancy. Germinal epithelium present as the outermost covering of ovary, beneath the germinal epithelium another layer is presently called as tunica albuginea, which is made up of connective tissue. The region of ovary under the tunica albuginea called ovarian cortex which contains fibroblasts, elastic fiber and collagen and all of them surrounded the ovarian follicles. Three major events which took place during the early stages of gonadogenesis responsible for the formation of a functional ovary: formation of follicles, differentiation of steroid-producing cells and initiation of meiosis.

The Follicle

An ovarian follicle contains different cell types which act as a complex unit. The mature preovulatory follicle contains layers of granulosa cells. Granulosa cells surround the oocyte and granulosa cells are surrounded by the layer of theca cell, layer of theca cell near the granulosa cell called theca internal and the layer of theca cell present outer side of theca internal called theca external. Granulosa cells are themselves containing specialized subpopulations of cells differ in their distribution of receptors and steroidogenic characteristics known as corona Radiata, cumulus cells, mural and antral granulosa cells. The cumulus cells surround the oocyte and nourish the oocyte as well contribute to the formation of the zona pellucida. Corona Radiata attached to cumulus and oocyte through gap junction forming an electrophysiological syncytium and corona Radiata maintain the close contact to oocyte through cytoplasmic extensions across the zona pellucida.

The granulosa cells which is nearby to the basement membrane (also called lamina propia present between theca and granulosa cell) are recognized as mural granulosa cells, while those nearby to the follicular antrum are known as antral granulosa cells. The basement membrane is an acellular layer contains fibronectin, laminin, proteoglycans and several types of collagen (collagen IV alpha 1 and alpha 2, reduced amounts of alpha 3-alpha 5).

The theca interna are the major source of androgens hormone. It is crucial for the final stage of development of the Graafian follicle. Follicles can be classified as primordial follicles, preantral follicles (primary and secondary follicles), antral and preovulatory follicles.

1.2.2.     Folliculogenesis

The development of ovulatory follicles throughout the reproductive life of a female at a fixed interval called folliculogenesis. The release of mature oocyte known as ovulation, ovulation take place in every 28 days.

Construction of the zona pellucida and the formation of product required for the fertilization and early embryonic development events take place during that extended phase. Follicles ultimately had two fates either ovulate as a mature oocyte or undergo atresia (atresia is known as the death of ovarian follicle), which can occur at any point during follicular development.

Primordial follicles

In newborn females small primordial follicles (fundamental developmental unit) are present and in the adult ovary, it is the prevailing follicle type. The concerned follicular epithelial cells were derived from the coelomic epithelium and when they combine with oocyte commonly known as a primordial follicle. Oocyte and follicular epithelium constitute the Primordial follicle. A single flat layer of follicular epithelium or granulosa cells or pregranulosa cell present on primordial follicle. This granulosa cells layer surround the oocyte.

Primordial follicles are positioned in the peripheral cortex of the ovary. As the follicles and oocyte start to grow, they move deeper into the cortex of the ovary.

Primary follicles

The primordial follicle increase in size proliferate. This stage is known as the primary follicle. The oocyte of the primary follicle is known as primary oocyte which enters into the first meiotic division, at this stage development of Follicular stimulating hormone (FSH receptor) also take place on primary follicle.

The primary oocyte is an arrest in the diplotene stage of prophase I. During the arrest different type of developmental changes occurs within the primary oocyte. The zona pellucida begins to form during the arrest of 1° oocyte. Oocyte not completely surrounded by the zona pellucida until the follicle reaches the late preantral stage. Groups of oocytes cyclically restart meiosis along with the onset of puberty. Thus the first part of meiosis begins in the embryo in human female and then arrest and wait for the signal to restart the meiosis, which comes at the onset of puberty roughly 12 years later. It is also a fact that some oocytes are upheld in meiotic prophase for nearly 50 years. At the time of birth, millions of primary oocytes present but only about 400 mature during a woman's reproductive life span.

Secondary Follicles

When puberty comes, the pituitary gland secretes the FSH which receives by the FSH receptor present on the primary follicle. In response to FSH the cell resume and complete the meiosis I and enter into meiosis II and the break of arrest occur and resume the meiosis takes place. At this stage 11 to 12 primary follicles are selected to further developmental process. The granulosa cells divide mitotically, the follicles are now called secondary follicles. The secondary follicles are bigger in size, has a new outer new outer layer of connective tissue, blood vessels, and theca cells. The cell work with the granulosa cells to produce estrogen. This primary follicle recruits the stroma like theca cells after oocyte signalling. The theca cells cover the granulosa outermost layer, basal lamina and differentiate the whole capsule into theca internal and theca external.

At this stage meiosis first get complete and the formation of secondary oocyte along a polar body takes place and chromosome no divide in half.

Tertiary or Antral Follicles

As the formation of antrum takes place and mark the formation of the tertiary follicle or (or late antral follicles). The antrum is a cavity in which follicular fluid get filled.

The granulosa cells first acquire the LH receptor. During the final stages of follicle maturation, the blood supply of the theca layer increases drastically.

Several follicles reach the tertiary stage at the same time, and most of these will undergo atresia.

The secondary oocyte or tertiary follicle having the largest size of the antrum and present nearby the periphery of ovary get selected to further developmental process at the seventh day of menstrual cycle and rest undergo atresia. Thus at eight days, only one secondary oocyte remain. This tertiary follicle grows further and called is Graafian follicle. The Ovulation takes place on the 14th day of the menstrual cycle. FSH stimulates the growth of a tertiary follicle, and LH stimulates the production of estrogen by granulosa and theca cells. Once the follicle is mature, it ruptures and releases the oocyte. Cells remaining in the follicle then develop into the corpus hemorrhagicum. Corpus hemorrhagium is a temporary structure, and within four days of ovulation, it is converted into corpus luteum. Progesterone is released by corpus luteum.

The trophoblast cell of blastocyst secretes human chorionic gonadotropin (hCG) hormones.  Human chorionic gonadotropin signals the corpus luteum to relax progesterone continuously. Corpus luteum is essential for the maintenance of pregnancy. It also releases relaxin hormones. Relaxin causes softening of pubic symphysis which helps in parturition (Delivery of the child). When there is no fertilization. The corpus luteum stops releasing progesterone after 10 days and converted into corpus albicans. When the egg is fertilized and implantation of the embryo occurs.

1.2.3.     Developmental competence

Oocyte ability to produce normal, viable and fertile offspring after fertilization. During follicular development, the acquisition of developmental competence is a gradual process.

The percentage of oocytes that can develop to the blastocyst stage usually expressed the developmental competence. Developmental competence also evaluates by morphological evaluations such as the number of blastomeres or the ratio between trophectoderm cell numbers and inner cell mass.

Apoptosis

Throughout embryonic and neonatal life apoptosis plays an important role in the massive loss of oogonia and oocytes.

The vast majority of follicular populations go through atresia rather than ovulation.

1.2.4.     Oocyte Maturation

Oocyte maturation takes place when oocyte progresses from the diplotene to the metaphase II stage and it is a complex process because nuclear maturation also takes place in this phase. The transition from the diplotene stage to metaphase is called diakinesis. In response to the ovulatory LH, surge oocyte resumes its meiosis or removal of the secondary oocyte from the follicle takes place. During diakinesis, folding of nuclear membrane get starts, the disappearance of nuclear pores occur and then the fragmentation of nuclear membrane occur before quickly disappearing to leave only small sacs with double walls and all these events are identified as germinal vesicle breakdown (GVBD), which is the first visible sign of meiotic continuation. When nucleolus comes in the contact with the cytoplasm get disappears.