Spermatogenesis

Question 1

Identify the main compartments of the testes, and state their main characteristics. How are these separated ?

Answer 1

1) Seminiferous tubules (intratubular compartment)
• ~250 m total length
• Developing germ cells
• Sertoli cells

2) Interstitial spaces (peritubular compartment)
• Leydig cells (synthesize androgens)
• Blood and lymph vessels

Both compartments separate: “Blood-testis barrier”

Question 2

How is the blood testis barrier formed ? What is the relation of this barrier with the spermatogenesis ?

Answer 2

Blood-testis barrier formed through interactions between tight junctions, zonular junctions, and filament bundles between neighboring Sertoli cells.

Spermatogonia residing on one side of this barrier, and
as development proceeds, primary spermatocyte transitions through blood testes barrier as it differentiates.

Question 3

What is the significance of the blood-testis barrier ?

Answer 3

• Prevents immune reaction to spermatozoa

* Separates fluids of different composition

Question 4

Define spermatogenesis. What are the main stages of it ?

Answer 4

Production of mature spermatozoa from undifferentiated germ cells (primordial germ cell). Occurs in three stages:

1) Mitotic proliferation (to generate exact copies of
sperm stem cells (so pool never depleted))
2) Meiotic divisions (Process creating female egg and male sperm cells. Reduces number of chromosomes in each sperm, so end up with 4 haploid spermatids for each primary spermatocyte generated)
3) Cell modeling (spermiogenesis) (remodels developing sperm cell into classic sperm shape)

Question 5

Outline the main steps of spermatogenesis.

Answer 5

MULTIPLICATION
1) Primordial germ cell undergoes mitosis and cell division, yielding type A and B spermatogonia (type A will yield more primordial germ cells whilst type B will continue in the development pathway), which changes neither the chromosome number in each cell (46), nor the DNA copy number (2C).

GROWTH
2) These spermatogonia then differentiate into primary spermatocytes (now 46, 4C). Each primary spermatocyte undergoes meiosis I and cell division, yielding two secondary spermatocyte. This divides the DNA content, and therefore changes both the DNA copy number (2C) and the chromosome number in each cell (23).

3) Each secondary spermatocyte undergoes meiosis II and cell division, yielding two spermatids. This is when double structures chromosomes separate, which changes the DNA copy number (1C) but keeps the chromosome number the same (23).

MATURATION
4) Spermatids are re-shaped, and then undergo the process of spermiogenesis to yield spermatozoa (which keeps both DNA copy number, and chromosome number the same, 23, 1C)
——
Spermiation then occurs.

Capacitation then occurs in the female reproductive tract, following ejaculation.

Question 6

How many spermatozoa are yielded for each spermatogonia entering meiosis 1 ?

Answer 6

4 spermatozoa for each spermatogonia that enters meiosis 1

Question 7

How long is the spermatogenic cycle ? What is the start and end point of the spermatogenic cycle ?

Answer 7

The Spermatogenic cycle is ~74 days (from 1st mitotic division to release of spermatozoa)

Question 8

How often does spermatogenesis occur ?

Answer 8

• Spermatogenesis occurs in waves, initiated every 16 days

Question 9

Describe the rate of spermatogenesis.

Answer 9

• Rate of each developmental stages is not uniform:

• Spermatogonium to primary spermatocytes = 25 days
• Meiotic division to secondary spermatocytes = 9 days
• Development to spermatids = 19 days
• Differentiation to spermatozoa = 21 days

Question 10

If you were to open a cross section of seminiferous tubule, how many layers with a different developmental stage would you expect to see ? Why ?

Answer 10

4 to 5, because 74 days for the whole cycle, divided by 16 (new cycle every 16 days), equals 4.6.

Question 11

Define spermiogenesis.

Answer 11

“That segment of spermatogenesis during which immature spermatids shed much of their cytoplasm and develop a flagellum and acrosomal granule, becoming, through this process, sperms.”

Question 12

How many sperm cells does the average man produced in a day ? in a second ?

Answer 12

About 200 million/day (Approx. 2300/second)

Question 13

Identify the main components of the final spermatozoon. Explain how the flagella moves.

Answer 13

• Most flagella (in end piece), like cilia, have a characteristic “9+2” structure, i.e., two central singlet microtubules are encircled by nine outer doublet microtubules. The outer and inner dynein arms slide along each outer doublet microtubule.
• In middle piece, sheaths of ring shaped mitochondria, providing energy for movement of the tail
• Principal piece has sheath around axoneme.
• Nucleus: repositioned and condensed into head piece. Formation of acrosome (formed by the Golgi, which produces a lot of enzymes). Enzymes in acrosome are then released on contact with ovum zona pellacida allowing sperm to make entry and fertilise the oocyte.

-Sliding is powered by flagellar dynein. This array of microtubules and associated motor and linker proteins is known as an axoneme. Mitochondria in the middle piece powers the conformational changes in flagellar dynein that mediate microtubule sliding

Question 14

Describe the state of the spermatozoa at the end of differentiation.

Answer 14

At end of differentiation:
• Cytoplasmic links are broken
• Spermatozoa released into tubule lumen
• Sperm virtually immobile

Question 15

Define spermiation. How does this occur ?

Answer 15

The release of mature spermatozoa from the Sertoli cells.

Fluid secreted by Sertoli cells flushes spermatozoa from seminiferous tubules, through the rete testis into the epididymis:
• Capacity for motility by the time they reach the tail of the epididymis
• Motility is suppressed by epididymal fluid
• Instead movement through reproductive tract is aided by peristaltic muscle contractions

Question 16

If ejactulated spermatozoa are placed with oocytes in vitro, what happens ? Why ?

Answer 16

If ejaculated spermatozoa are placed with oocytes in vitro fertilization does not occur immediately.
They need to undergo the process known as capacitation.

Question 17

Describe the process of capacitation.

Answer 17

• Normally occurs in the female reproductive tract (2-6h)
• Glycoprotein coat gained in the epididymis is stripped
• This results in two changes:
- Head acquires the capacity to initiate the acrosome reaction
- Hyperactivation (increased flagellar beats)

Question 18

What proportion of human sperm show normal morphology under a microscope ?

Answer 18

Only 4-14% of sperm show normal morphology under a microscope

Question 19

Under what proportion of normal morphology sperm under the microscope are fertilization rates obtained with IVF reduced ?

Answer 19

If normal morphology drops below 4% fertilization rates obtained with IVF are reduced.

Question 20

How is infertility in men diagnosed ?

Answer 20

Infertility in men is primarily diagnosed by semen analysis comprising of:
• Determination of sperm concentration/total count
• Motility
• Morphology

Question 21

Identify the main semen parameters, and state their reference range.

Answer 21

Semen volume (>1.5 ml)
pH (>7.2)
Sperm concentration (>15 million sperm/ml)
Total sperm count (>39 million sperm/ejaculate)
Total sperm motility (>40% motile sperm)
Sperm morphology (>4% morphologically normal sperm

Question 22

Identify and define the main types of sperm abnormalities.

Answer 22

Oligozoospermia
• Reduced sperm count (< 15 million/ml)

Azoospermia
• Absence of sperm in the ejaculate

Asthenozoospermia
• Reduced sperm motility (< 40% moving)

Teratozoospermia
• Reduced percentage of sperm with normal morphology

Antisperm
• Abnormal immune response to sperm

– *Combinations of these abnormalities are common

Question 23

Describe the role of the hypothalamus and pituitary gland in spermatogenesis.

Answer 23

Secretion of hormones by the hypothalamus, pituitary and testes are regulated by complex feedforward and feedback loops

Pulsatile secretion of Gonadotrophin- releasing hormone (GnRH) from the hypothalamus stimulates the anterior pituitary gonadotrophs

The pituitary gonadotrophins, LH and FSH, control testicular function (including the regulation of steroidogenesis and sperm cell maturation)

Question 24

Identify the main functions of FSH and LH. Which cells do they respectively act on ?

Answer 24

Leydig cells in the testes have receptors for LH
– stimulates synthesis and secretion of testosterone
(BUT LH and testosterone cannot maintain spermatogenesis at its normal level without FSH)

FSH is required for maximum sperm production
– acts on Sertoli cells

Question 25

State the effect of low LH on spermatogenesis.

Answer 25

If LH secretion is too low, testosterone is low

– spermatogenesis halts

Question 26

Describe the hormonal cascade between the hypothalamus, pituitary and gonads.

Answer 26

• GnRH produced in pulses, stimulating FSH and LH
• FSH (from anterior pituitary) target FSH receptors on sertoli cells, and induces increased amount of aromatase
• LH (from anterior pituitary) targets its receptor on leydig cells and stimulates synthesis of testosterone
• testosterone freely diffuses into sertoli cells without even crossing blood-testes barrier
• In sertoli cells, testosterone can promote expression of androgen-binding protein, which will act to trap testosterone in higher concentrations
• Aromatase converts testosterone into estrogen, which is produced by sertoli cells in the male
• Estrogen has local action in testes and stimulates production of inhibin which feeds back to anterior pituitatry in negative way and inhibits the production and secretion of FSH
• Testosterone, in the Leydig cell, can be converted into its more active metabolite, DHT, or can also circulate back to both hypothalamus and pituitary and decrease LH and FSH secretion, or further upstream to decrease secretion of GnRH

Question 27

What does low inhibin indicate relative to spermatogenesis ? Why ? How does it affect gonadropin levels ?

Answer 27

One of indicators of decreased spermatogenesis is decreased inhibin (normally, will feedback and regulate homeostatic function but if low spermatogenesis, will not produce inhibin).

Also, excess FSH in blood because no negative feedback loop)

Question 28

What does low testosterone indicate relative to spermatogenesis ? Why ?

Answer 28

If have decreased testosterone, testosterone itself will not negatively feedback, and so this will increase gonadropins levels (FSH and LH).

Question 29

Identify the main functions of FSH on Sertoli cells.

Answer 29

FSH activates various pathways, including:

• ↑ RNA and protein synthesis
• ↑ Energy metabolism
• ↑ Inhibin secretion
• ↑ Cyclic AMP
• ↑ ABP secretion
• ↑ Fluid secretion (helps to flush developing sperm into rete testis)
• ↑ Androgen receptors which increases FSH receptors (feedforward loop)

Question 30

Describe the result of a loss in FSH, and LH receptor respectively on spermatogenesis. Consequently, which is the key hormone here ?

Answer 30

1) Loss of LH receptor function results in severe decrease in testosterone which results in complete lack of spermatogenesis.

Loss of FSH receptor, reduced capacity of sperm production, but still some.

Hence, key hormone driving process is LH which stimulates testosterone which is critical to support Sertoli cells.

Question 31

Identify the main hormones produced by the testes (and state the specific type of cell which produces each hormone).

Answer 31

1) Testosterone (steroid) by Leydig cells
- In some tissues do not exert direct effects, but are converted to dihydrotestoterone (DHT) or estrogens (through aromatisation)

2) Estrogens (steroid) by Sertoli and Leydig cells

3) Inhibins (peptide hormone) by Sertoli cells
- feedback loop to control hormone levels (at level of pituitary)

4) Oxytocin (peptide hormone) by Leydig cells
• contraction of smooth muscle of the genital tract

Question 32

Describe the role of testosterone in spermatogenesis.

Answer 32

Essential for spermatogenesis
– If production prevented spermatogenesis ceases
– If production decreases (i.e. blood [testosterone[ is low) then fewer stem cells begin cell division but the whole process will still take 74 days.
– Blocked when primary spermatocytes enter meiotic prophase

Question 33

What is the effect of too high testosterone on spermatogenesis ? Why ?

Answer 33

Too high testosterone will also reduce spermatogenesis.
Too much generates a lot of estrogen locally, which will produce a lot of inhibin, which will have a negative feedback.

(but high testosterone increases libido despite low sperm count)

Question 34

What proportion of infertile men receive a causal diagnosis ?

Answer 34

Only ~28% of infertile men are provided a causal diagnosis
~72% of men in infertile couples, no causal diagnoses can be established and the aetiology of disturbed spermatogenesis remains largely unclear.

Question 35

What causal diagnoses are the men who receive a causal diagnosis given ?

Answer 35

– Most consist of previous gonadotoxic chemo- or radio- therapy for the treatment of malignant disease such as testicular cancer (~10%)
– Testosterone abuse or other chronic diseases (e.g. diabetes) (~14%)
– Genetic disorders (e.g. Klinefelter syndrome, Karyotype 47, XXY or microdeletions on the Y-chromosome (4%))
– In addition, genes involved in the migration and function of GnRH neurons or their upstream regulators have been discovered that may be mutated in patients with congenital hypogonadotropic hypogonadism with or without anosmia (lower circulating gonadotropins and correspondingly low gonadal function)