Riassunto esame Embriologia, Prof. Castagneti Lara, libro consigliato Carlson, Bruce M. Carlson

Introduction to Embryology

Purposes of studying embryology

Studying embryology helps doctors and humans in many ways:

• Helps to understand how the human body develops
• Helps illuminating serious clinical situations (such as cancer or physical disability/condition, that may arise from mutations or genes re-activation that happen during body development)

Periods of human embryology

There are two ways to divide the period of human development: a medical and an embryological one.

• Medical = 3 trimesters
  • Period of the egg: from fertilization to implantation (zygote → morula → blastocyst)
  • Period of the embryo: from implantation (1^st week) to 8^th week
  • Period of the fetus: from the 8^th week to the end of pregnancy and birth

Dating pregnancy

Pregnancy can be dated through different criteria, starting either from the fertilization date or the onset of the last menstrual period.

• Fertilization age: This method is very precise but almost impossible to use. Indeed, the exact moment in which the spermatozoa enter the egg should be used for counting (= difficult because it can survive up to 4 days while the egg up to 2 days). With this calculation the pregnancy lasts 38 weeks. Pregnancy is divided into the period of early embryo, period of embryonic organogenesis and fetal period.
• Menstrual age: This method is commonly used in clinical evaluations; the counting starts on the onset of the last period. Since we can’t be sure of the precise moment of the fertilization, we add 2 weeks of uncertainty: the total is 40 weeks.

Teratogens

They are agents (chemical, physical or biological) that alter fetal morphology or functionality (if the fetus is exposed during critical times). In relation to teratogens, embryo development can be divided in three phases:

• Resistant period (weeks 1 and 2) → the embryo either remain unaffected or dies
• Maximum susceptibility period (weeks 3 – 8) → teratogens affect organ development
• Lower susceptibility period (weeks 9 – 38) → as organs have already been planned and arranged, changes and possible defects will not be macroscopically visible (however, there may be functional derangements)

Causes of birth defects

They may be classified in three main categories:

• Genetics: chromosomal abnormalities (in number or structure, such as deletions and translocations for chromosomes or punctiform mutations for single genes)
• Environmental factors: drugs, chemicals, viruses or radiations
• Multifactorial inheritance (20-25%): predisposition of genetic and environmental factors that act together
• Unknown etiology (50-60%)

Body axes and planes

There are two ways to describe the human body:

• Anterior/posterior for adults – ventral/dorsal for embryos
• Superior/inferior for adults – cranial/rostral or caudal for embryos

Embryos can also be studied from transverse sections, median or sagittal, coronal or frontal sections.

Overview of Genital Tracts

Male genital tract

The major components are:

• Penis
• Epididymis
• Testes (origin of spermatozoa in seminiferous tubules, then they are carried to the epididymis and ductus deferens)
• Ductus deferens
• Scrotal bursa (where testis and epididymis are contained)
• Sperm vesicles
• Prostate gland
• Urethra (in common with the last part of the urinary tract, receives the spermatozoa via rete testis and ductus deferens)

Female genital tract

It is composed by:

• Ovaries (where the oocytes mature every month)
• Fallopian tubes (the tube where the mature oocyte goes and may encounter fertilization)
• Uterus (it can be the site for implantation for the fertilized cell, or its inner membrane, the endometrium, can break every month if fertilization doesn’t happen)
• Vagina
• External genitalia (vulva)

Phases of Human Embryology

The phases are:

• Gametogenesis: formation of gametes
• Fertilization: fusion of male and female gametes (zygote)
• Cleavage: proliferation of cells in zygote
• Gastrulation: formation of the 3 basic layers from which all embryo tissue develops
• Formation of body plan (morphogenesis): from a flat object (initial embryo), it folds to form tubular structures (tubes-within-tubes), eventually becoming a complex curved object with rudimental organs
• Organogenesis: formation of internal organs from the 3 embryological layers

Introduction to Gametogenesis

Cells that give rise to gametes (primordial germ cells, PGCs) reside in the yolk sac (extraembryonic membrane) and probably arise during the phase of gastrulation. Their lineage constitutes the germline, and they can be clearly identified for their characteristics (pale cytoplasm, rounded shape, and other molecular markers).

• Primordial cells migrate into dorsal body wall.
  They migrate by ameboid movement (they move through cytoplasmic adhesion = lamellipodia, actin filaments) from the yolk sac to the gut tube (the primitive precursor of both the respiratory and digestive systems) and populate the region of the body wall at the level that will form the gonads. They continue to multiply by mitosis; some may become stranded (= teratoma). Tumors of tissues derived from all germ layers (extragonadal or gonadal). Most common tumors in newborns are formed by pluripotent cells (able to form a variety of anatomic structures like eyes).

PGCs arrive in the gonad region (or genital ridge) and stimulate the epithelial cells to form somatic support cells which create swellings that will become gonads. Somatic support cells give rise to:

• Ovarian follicles (female)
• Sertoli cells of the germinal epithelium of the seminiferous tubules (male), which will assemble into testis cords.

Gametogenesis, Fertilization, and First Week

Gametogenesis

The timing is different in men and women.

• In males: PGCs remain dormant from the 6^th week of embryonic development until puberty (seminiferous tubules mature and host cells that start will differentiate through spermiogenesis, spermatozoa are then produced until death)
• In females: PGCs undergo more mitotic divisions after becoming invested by somatic support cells; then they differentiate into oogonia. By the 5^th month of fetal development all oogonia begin undergoing meiosis (they become primary oocytes). After, all sex cells enter a state of dormancy and remain in mitotic arrest until sexual maturity. At puberty each month a few ovarian follicles resume development in response to the monthly surge of pituitary gonadotropic hormones, but usually only one primary oocyte matures into secondary oocyte and is ovulated. This oocyte enters a second phase of meiotic arrest and doesn’t complete meiosis unless it is fertilized.

Function: PGCs contain 23 pairs of chromosomes or 46 chromosomes; one pair comes from the maternal gamete and the other from the paternal. The first 22 pairs are called autosomes while the remaining two are sex chromosomes. Somatic cells and PGCs are diploid (2n) while mature gametes have only one copy of each kind of chromosome and are called haploid (n); after duplication (before mitosis and meiosis) the cells are 4n.

In meiosis, a diploid cell (2n) replicates its DNA (becoming 4n) and undergoes two successive, qualitatively different nuclear and cell divisions to yield four haploid (1n) offspring.

Meiosis with germ cells

In the female, the oogonium is now a primary oocyte while in the male the spermatogonium is now a primary spermatocyte. Once the DNA replicates, each chromosome consists of two parallel strands (chromatids) joined together in a region called centromere.

Stages of meiosis I

• Preparatory phase: DNA replication yields double-stranded chromosomes
• Prophase 1: chromosomes condense; two chromosomes of each homologous pair align at the centromeres to form a chiasma (eventual crossing over)
• Metaphase 1: chromosomes align along the equator
• Anaphase and telophase 1: each double-stranded chromosome of each homologous pair is distributed to each daughter cell
• Cytokinesis: cell divides (result: two secondary spermatocytes, one secondary oocyte and one polar body; haploid 2n)

Stages of meiosis II

• Prophase: double-stranded chromosomes condense
• Metaphase: chromosomes align along the equator and centromeres replicate
• Anaphase and telophase: each chromosome splits into single-stranded chromosomes, one of which is distributed to each daughter nucleus
• Cytokinesis: cell divides (result: four spermatids, one definitive oocyte and three polar bodies; haploid 1n)

Spermatogenesis

Puberty: Testes begin to secrete great amounts of testosterone (steroid hormone), which has the following functions: development of sex characteristics, triggering testes growth, maturation of seminiferous tubules and commencement of spermatogenesis. Sertoli cells then differentiate into a system of seminiferous tubules. PGCs resume growth and differentiate into spermatogonia, they are located under the basement membrane surrounding the seminiferous tubules (pockets between Sertoli cells). They also are interconnected by tight junctions which help establish a blood-testis barrier developing an immune privileged site during their development in the testes. This translocation from the basal to the luminal side of seminiferous epithelium takes place during spermatogenesis and the final process of sperm cell differentiation (maturation of spermatids into spermatozoa) is called spermiogenesis.

Spermatozoon: head (condensed nucleus and cap of apical vesicle, acrosome, containing hydrolytic enzymes) + midpiece (large mitochondria for swimming) + tail (microtubules that form the propulsion system).

Male hormones

They are the same as the female’s: FSH (follicle-stimulating hormone) and LH (luteinizing hormone).

1. FSH acts on Sertoli cells (wall of seminiferous tubules)
2. LH stimulates Leydig cells, which then produce testosterone.

Function of Sertoli cells

They are somatic cells of the testes and participate in the process of spermatogenesis in many ways:

• They form a blood-testis barrier: creation of a basal compartment (separated from the luminal) where spermatocytes can grow, as they are antigenically different haploid germ cells.
• They secrete the tubular fluid inside the seminiferous tubules
• They produce an androgen-binding protein, and many other proteins, such as the retinal-binding proteins, Mullerian-inhibiting factors, and inhibin for feedback loop to hypothalamus (it goes back to the hypothalamus so that less hypothalamic-releasing factor is produced, stimulating the production of the follicle-stimulating hormone)
• Maintenance and coordination of spermatogenesis
  • Their cytoplasmic processes embrace spermatocytes as they go through different stages of maturation
  • They are responsible for making sure spermatogenesis proceeds in the right way.

Waves of spermatogenesis in a seminiferous tubule

Although spermatogenesis occurs continuously and gametes are produced in synchronous waves, in each local area of the germinal epithelium the process is not synchronized throughout the seminiferous tubules (about 4 waves can be observed in any region of the tubule at any time). A spermatogenic wave is called a cycle (we see a homogeneity in the stage of maturation); it takes more or less 64 days to find a new wave in the same segment.

Sertoli cells fundamental in spermiogenesis

Maturing spermatocytes and spermatids are connected to surrounding Sertoli cells by intercellular junctions and cytoplasmic processes called tubulobulbar complexes. As the cytoplasm of developing gametes shrinks significantly during spermiogenesis, the tubulobulbar complexes help transfer the excess cytoplasm to Sertoli cells. The process of spermiation happens when the last connection between spermatozoa and Sertoli cells break, and they are released in the lumen. By losing the cytoplasm they develop a tail.

The events during spermiogenesis can be divided into two categories:

• Nuclear events: change in the shape and size of nucleus, obtained by condensing chromosomes (replacement of proteins – histones – that normally compact DNA with protamines, that compact DNA even more)
• Cytoplasmic events:
  • Elimination of some cytoplasm (size shrinking) to make spermatids lighter (through phagocytation from Sertoli cells)
  • Formation of a head through condensation of Golgi apparatus at the apical end of the nucleus (the acrosome, filled with proteolytic enzymes to perforate the egg membrane and penetrate the cell during fertilization)
  • Formation of a tail (flagellum) on the other side of the centrioles with respect to the head (for spermatozoa motility)
  • Spiral arrangement of the mitochondria in the proximal flagellum for energy production

Results

The membrane of the head of the spermatozoa becomes antigenically different and is divided into different antigenic domains (changing during maturation in the male and female genital tract). The total length of the head and tail combined is 60 micrometers.

The cytoskeleton of the tail is the axonema, made of microtubules (in particular, 9 pairs of microtubules disposed in a circle and 1 central pair; each pair is made of an A-microtubule and a B-microtubule). The protein dynein helps spermatozoa with their overall motility: when microtubules are bending, dynein is active and attached to them.

The cilia in the ductus deferens help the circulation of spermatozoa; sometimes they lose their ability to be motile (ciliopathy), or have deficiency in the axonemal structure, leading to infertility.

Functional maturation of spermatozoa

When released in the lumen of the seminiferous tubules, spermatozoa are pushed by the fluid’s pressure, muscle contraction, and ciliary movements, towards the rete testis, the efferent ducts, and the epididymis.

Sperm cells are stored in the lower part of the epididymis (4.5-6 meters coiled duct connected to vas deferens) for about 12 days. The acquisition of motility takes place in the epididymis as their cells produce the factor motility protein, which induces within the spermatozoa:

• Tubulin phosphorylation
• Increase of intracellular concentration of calcium
• Increase in cyclic AMP (cAMP, a second messenger that easily passes through cell membranes)

In the epididymis, spermatozoa also undergo some biochemical changes: the head is coated by glycoproteins, needed for protection during the journey towards the egg.

During ejaculation, sperm are propelled through the vas deferens and urethra and are mixed with nourishing secretions from the seminal vesicles, prostate, and bulbourethral glands. Many spermatozoa may be deposited in the vagina in a single ejaculation, but only a few hundred navigate through the cervix, uterus, oviduct, and ampulla region. In the ampulla, sperm survive and retain their capacity to fertilize an oocyte for 1-3 days.

Capacitation reaction

The final step of spermatozoa maturation is the capacitation reaction: a series of changes in the acrosome that allow it to release enzymes and penetrate the zona pellucida (shell of glycoproteins around the oocyte). Location: within the female genital tract (requires contact with oviduct’s secretions).

Freshly ejaculated sperm is unable or poorly able to fertilize so they must undergo a series of reactions for capacitation. Capacitation is necessary for the future acrosomal reaction (spermatozoa enzymes will digest the membrane surrounding the oocyte); it consists in the removal of plasma membrane proteins and reorganization of lipids and proteins. Result: changes in antigenic domains and glycoproteins are mostly removed.

The final “product” is called semen (the ejaculate). Its volume goes from 2 to 6 ml circa and it is composed of sperm (less than 10%, around 100,000,000 spermatozoa/ml) and secretion of prostate glands and seminal vesicles.

Causes of male infertility

Detectable in 30-50% of involuntary childless couples include:

• Reduced number of spermatozoa (less than 10,000,000/ml)
• Lack of motility
• Abnormal sperm (e.g., sperm with double head or tail)
• Genomic alterations
• Medications or drugs (they could go through the blood-testis barrier)
• Endocrine disorders
• Environmental pollutants
• Smoking
• Obstruction of genital duct system

Oogenesis

By 12 weeks of development, oogonia in genital ridges enter the first meiotic prophase and almost immediately become dormant; the nucleus becomes large and watery.