The Testis
Testis 1 - Dr. Sakkas
Development of the Testis
Gonads are bipotential for first 7 weeks of gestation
Y chromosome is essential for sex determination and gonadal differentiation
SRY portion is the genetic determinant of maleness
SRY gene encodes for Testis Determining Factor (TDF) that promotes the Mullerian Inhibiting Factor (MIF; AMH)
Ovary is default pathway when TDF is not present
Spermatogenesis
Sperm are released into the seminiferous tubule to go into the vas deferens and epididymis
Dramatic change in shape and form during period of spermatogenesis
Sertoli cells line the walls of seminiferous tubules from the basal portion to the lumen
Sertoli cells are the framework of seminiferous tubules
Developing sperm migrate from the basal layer to the lumen to then be released into the vas deferens and epididymis
Seminiferous tubules coordinate spermatogenesis
Key Cells in the Testis
Sertoli Cells
Forms the framework of seminiferous tubules
Testicular equivalent to granulosa cells
Produces inhibin to regulate FSH production
Support and protect spermatogonia, phagocytize degenerating spermatogonia/spermatocytes
Produce nutritive fluids such as proteins, enzymes, RNA, and ions
Constantly talking to germ cells to regulate function of those germ cells through their development
What you have up until puberty is what you end up with → no stem line for Sertoli Cells
Forms blood-testis barrier
Leydig Cells
Interstitial endocrinocytes
Located between seminiferous tubules that are vital for testosterone production
Not many, important for their function
Germ Cells
Spermatogonia
Blood-Testis Barrier
Sertoli cells have specialized tight junction
Spermatogonia and early spermatocytes lie outside of these tight junctions and the remaining germ cells lie inside the testis-tubule barrier (delineation of spermatogenesis as sperm develop that protects the sperm as they are developing)
This barrier acts to exclude potentially damaging agents such as antibodies and toxins
Endocrine Function
Leydig cells are primarily responsible for the majority of androgen production in the body
95% of testosterone circulating in the male bloodstream originates from Leydig cells
Male fertility depends on the endocrine and paracrine functions of androgen produced by the testes via the hypothalamo-pituitary-testicular axis
Key Hormones
LHRH (LH releasing hormone)
LH
FSH
Testosterone
Inhibin
Feedback Mechanisms
Where the action of a hormone increases/decreases the effect of another hormone on itself
LH stimulates Leydig cells to produce testosterone and testosterone levels regulated via negative feedback mechanism
FSH stimulates Sertoli cells to produce inhibin and inhibin levels regulated via negative feedback mechanism
Testosterone
Produced by Leydig cells, regulated by LH
After birth, androgen secretion remains low until puberty, while testosterone secretion recommences in response to increased stimulation by LH
Is secreted in a pulsatile manner in response to pulsatile LH released by the pituitary gland
Circulating testosterone acts via androgen receptors located in target cells to stimulate secondary characteristics and exert negative feedback regulation of LH secretion
Free testosterone: circulates in the plasma
In the blood, unbound to binding proteins
Targets
Vocal cords: voice deepening
Skin and fair follicles → hair on arms, legs, face
Bones: get stronger
Skeletal muscles: get stronger
Central Nervous System: different reactions could relate back to hormonal activity
Inhibin
Exerts a negative feedback regulation of FSH secretion before puberty
Putting a brake on FSH secretion
Along with testosterone, participates in the homeostatic control of circulating plasma FSH levels
Spermatogenesis
Complex process of proliferation and differentiation transforming spermatogonia into mature spermatozoa
Involves a series of mitosis and meiosis and changes in both cytoplasmic and nuclear architecture
Occurs to control the shape change of sperm
Outcome of spermatogenesis affected by the extent of programmed cell death
Apoptosis attempts to control numbers and quality of sperm produced
Mitosis
Increases cell numbers by division
End with two identical cells
Start with diploid, end with diploid
One cell will replenish the pool for spermatogensis later on, while the other undergoes spermatogenesis (spermatocyte development)
Spermatogonia are diploid (2N = 46) at base of tubule (basal side)
Cell division produces more diploid spermatogonia
Spermatogonia (2N) become primary spermatocytes
The spermatogonia that is dedicated to to spermatogenesis is termed spermatogonia Tybe B
Meiosis
Halves the chromosome number and generates genetic diversity
1 primary spermatocyte (2N) divides to produce 4 haploid (N = 23) spermatids
Spermatids all equal in size and very small
During first round of meiosis (M1) centromeres do not divide (2n)
During M2, centromeres divide, result in N
Testis I
Intro
Mobile, particular shape, head and tail
All cellular events that occur to make a cell are occurring in sperm
In mammalian species, sperm production occurs at a lower than core body temperature, more efficient, many males have low hanging / positioning of balls
Development of the Testis
Gonads are bipotential for the first 7 weeks → no differential between the tissues
1959: Y chromosome in mammals was essential for development as a male
1991: Small fragment of the Y chromosome (SRY) was shown to be the genetic determinant of maleness
Encodes for the Testis Determining Factor
TDF: protein that promotes the Mullerian Inhibiting Factor (AMH/MIF/MIS)
TDF Absent = Ovary
TDF Present = Testis → testosterone and MIF production
Ducts
Default mechanism = Mullerian ducts, fallopian tubes, uterus
With SRY Gene activation, MIF, and TDF
Mullerian ducts are inhibited, and broken down. Wolffian duct go on to form the epididymis and vas deferens
Epididymus wraps around testis, vas deferens
Seminal plasma and seminal vesicle prostate gland
Sperm-producing tube have rings of tubules (seminiferous tubules within testis), that lead to the epididymis and vas deferens, which pools into the seminal vesicle, prostate gland
In all seminiferous tubules → spermatogenesis occurs from the basal area into the tubule area
Lumenal area, basal area, interstitial area between seminiferous tubules
Larger/mature cells and nuclei from the outside of the tubule
Development
Spermatogonia → spermatocytes → early/round spermatids → elongated spermatids → mature spermatozoa/sperm
Must be coordinated in hormonal and cell function fashion
Once sperm matures, it is released into the vas deferens and epididymis
Sertoli Cell: goes from the lumenal to basal wall, framework of the seminiferous tubules
Seminiferous tubules help coordinate spermatogenesis → happens along the walls of the Sertoli cells
A lot of connective tissue around the seminiferous tubules, particular cells between the tubules that are important for testosterone production
Key Cells in the Testis
Leydig Cells: reside outside/between the seminiferous tubules, interstitial endocrinocytes
Key cells for producing hormones (testosterone)
Constitute less than 1% of the total testicular mass
Originate from 2 sources → differentiation from mesenchymal cells and from the division of Leydig cells under the influence of LH and FSH
Sertoli Cells: testicular equivalent to ovarian granulosa cells, sperm producing cells
Produce inhibin
Feed and regulate spermatogenesis along the testicular seminiferous wall
Support/protect spermatogonia, phagocytize (bit of a macrophage) degenerating spermatogonia / spermatocytes
Produce nutritive (protein, enzyme, ions) fluids and feed sperm
Do not divide in the adult testis, no extra Sertoli cells developing post-puberty
Form blood-testes barrier: sperm are antigenic
→ don’t want to create a cell that can be phagocytosed by other cells, protects sperm when entering female reproductive tract so they won’t be seen as foreign enemies
Sertoli cells have specialized, tight junctions
Spermatogonia and early spermatocytes lie outside and the remaining germ cells lie inside the testis-tubule barrier
This barrier acts to exclude potentially damaging agents such as antibodies and toxins
Important the tight junction is intact
In constant contact with germ cells to regulate function/development of sperm
Endocrine Function
Occurs in the compartment between the tubules
Leydig cells are primarily responsible for the majority of androgen production in the body
95% of the testosterone circulating in the blood originates from Leydig Cells
Although there are few of them, very integral to function
Hypothalamus-Pituitary-Testicular Axis (HPT)
Male fertility depends on the endocrine and paracrine functions of androgens produced by the testes
Hormones
Reproductive messenger throughout the body, particular in how they deliver their message
Steroids, proteins, glycoproteins, or peptides
All hormones, regardless of structure, work through receptors that are normally outside the cell but can also be intracellular
Nuclear receptors exist for some hormones
The chemical structure of the hormone determines how and which receptor it activates
Lock and key theory → cells contain specialized receptors
LHRH: Luteinizing hormone-releasing hormone
LH: Luteinizing hormone FSH: Follicle Stimulating Hormone
Testosterone Inhibin
Feedback mechanism: action of a hormone increases or decreases the effect of another hormone or itself
AXIS EXPLAINED
The hypothalamus controls the posterior and anterior pituitary
FSH will trigger a response from the Sertoli cell
Inhibin will inhibit hypothalamus activity, looping back
LH will trigger a response from the Leydig cell, inhibiting testosterone production by sending negative feedback to the hypothalamus.
Will produce testosterone to activate Sertoli cell, but also alert the hypothalamus to halt production
Testosterone
Produced by the Leydig cells and regulated by LH (feedback mechanism)
Activated by maternal HCG during fetal and neonatal life
HCG and LH are very similar, can share receptors, combined to each others receptors
After birth, androgen secretion remains low until puberty, when testosterone secretion recommences in response to increase stimulation by LH → puberty
Is secreted in a pulsatile manner in response to pulsatile LH released by the pituitary gland
Pulsatile manner is more efficient in regulating hormonal activity and the feedback mechanisms
Biosynthesized from cholesterol, obtained from blood-borne high-density lipoproteins
Circulating testosterone acts via androgen receptors located in target cells to stimulate secondary characteristics and exert negative feedback regulation of LH secretion
Responsive target cells: vocal cords, skin/hair follicles, bones, skeletal muscles, central nervous system (brain)
Can be used to explain differences in metabolism, brain function → attributed to hormones
Binding proteins bind remainder of the testosterone circulating in your body
Free testosterone is what is important
Inhibin
Exerts a negative feedback regulation of FSH secretion before puberty
Putting a brake on FSH, Sertoli Cell
Along with testosterone, participated in the homeostasis control of circulating plasma FSH levels
Hypogonadotropic Hypogonadism: low testosterone with LH and FSH
Spermatogenesis
Complex process of proliferation and differentiation transforming spermatogonia (circular) into mature spermatozoa (small head long tail)
Unique process involves a series of mitoses and meioses and changes in both cytoplasmic and nuclear architecture
Outcome of spermatogenesis is affected by the extent of programmed cell death (apoptosis)
Control #/quality of sperm produced through apoptosis
Spermatocytes, just outside the blood-testes barrier, delineation of the sperm germ cells that squeeze through tight junctions and become early spermatids
Mitosis in the spermatogonia
Meiosis occurring in spermatocytes to round spermatids
Differentiation in the elongated spermatids to the mature spermatozoa
Sertoli cells creating concert of exchange that allows spermatogonia to progress to mature spermatozoa
Tight Junctions
Series of protein that form the zip-like structure between cells
Preclude anything from passing between those cells, can control
Cells can squeeze through, closed above it and open below it to pass through
Kinetics of Spermatogenesis
Man → 64 days Bull → 54 days Ram → 49 days Boar → 34 days Rat → 48 days
Mitosis
Increases cell numbers by division
Dedicate one cell to the spermatogenesis pathway and retain one cell for spermatogenesis later on in life
Duplicated cells
Cell division, chromosome replication, and chromosome segregation
One will replenish the pool of spermatogenesis while other will go through the process of maturation
Spermatogonia (diploid) (2n=46). At base of tubule
Cell division produces more diploid spermatogonia
Spermatogonia (2N) become primary spermatocytes (2N)
Spermatogonia type A1 → 1 of them becomes spermatogonia type B → becomes spermatocyt
Meiosis
Halves the chromosome number and generates genetic diversity
1 primary spermatocyte (2n) divides to produce 4 haploid (N=23) spermatids
Spermatids all equal in size and very small
Secondary spermatocyte goes through M2 to produce round spermatid
Start with 1 spermatocyte, end with 4
In M1 → centromeres do not divide, maintaining 2N
Telophase: 2N, same # of chromosomes
M2 → centromeres divie, all four have 1 N
Interphase II: division, separating into true haploid cells
Testis II - Dr. Sakkas
Sex Chromosomes
Male sperm brings the sex capabilities to the oocyte for sex determination of the offspring
Oocyte will always contribute an X while sperm can contribute X or Y
Spermiogenesis
Final stage of spematogenesis
Spermatids develop morphological traits of a sperm cell
All changes that take place during spermiogenesis are accomplished without any new gene expression or protein synthesis, since spermatids lack transcription and translation machinery
No further changes in nuclear consistency (remains as a haploid cell), but changes in the shape to a more streamlined cell to better suit motility
Tail structure and nuclear compaction to create smaller head
Once the nucleus is heavily compacted, DNA replication, transcription, and translation are suppressed
DNA Packaging
Histone packaging to create nucleosomes that form chromosome
Protamine
In sperm DNA packaging, the histones are taken away during spermiogensis and replaced with the nuclear protein called protamine
Protamine creates a tight packaging system to promote smaller head and tail of sperm
Improper DNA packaging can result in inheritance errors (epigenetic, RNA modification)
Regions of Mature Spermatozoa
Acrosome (cap): holds digestive enzymes for fertilization
Outer membrane to the head
Has various receptors that bind during the process of fertilization
Head (nucleus): stores DNA
Mid-piece (mitochondria): source of ATP
Provides energy to get the tail to move
Flagellum (tail): locomotion
Loss of Cytoplasm
The Sertoli cell has a macrophage-type property with a continual cross-talk with the sperm cells
Sperm develop along the walls of the Sertoli cells
Large plastic droplets are starting to be sucked into the Sertoli cells
Shape of the sperm is being formed
Sertoli cells are acting like macrophages by sucking in part of the cells
Extra cytoplasm is sucked in by the Sertoli
Male Infertility
Recognized as the single most common cause of infertility, with sperm defects accounting for around 30-50% of cases presenting to fertility clinics
Semen analysis: prepare sperm, looking for motility count, and shape of sperm
WHO: reference values of male infertility
Sperm numbers < 15 million/mL
Total motility < 40% motile
Morphology < 15% normal shape
Combinations of problems
Oligozoospermia: low numbers of sperm
Asthenozoospermia: poor motility
Types
Azoospermia: no sperm presence of sperm in ejaculatory duct
Obstructive Azoospermia: Testis functions properly to make sperm, but there is a “road block” in terms of allowing the sperm out
Easy to treat by surgically removing sperm
Non-Obstructive Azoospermia
Two most common categories of genetic factors associated with non-obstructive azoospermia: Chromosomal abnormalities + Y-Chromosomal microdeletions
Spermatogonia present but no progress in spermatogenesis
Many patients opt for sperm donors
Intracytoplasmic Sperm Injection
Pioneered in 1992
Sperm is taken up in a pipette → oocyte
Tapping sperm tail renders sperm immotile, won’t run away
Must break cytoplasm of the oocyte for efficient injection of sperm
Only need one sperm per oocyte → efficient
DNA Damage in Sperm and Aging
Increased paternal age associated with adverse outcomes for offspring
Live pregnancy rate declines
Potential association with autism
Key Process
Acquisition of the sex chromosome
Oocyte will carry x and sperm will carry x or y
In addition to adding genetic diversity during meiosis, you also have allocation in the haploid cells of x or y, brings sex capabilities to the oocyte
Fluorescence in Situ Hybridization
Old tech used to look at chromosomes in sperm
Meiosis in spermatogenesis is quite efficient in comparison to the oocyte
Spermiogenesis
Final stage of spermatogenesis (differentiation phase)
Further maturation
Develops morphological traits of a sperm cell
No growth phase, lose a lot of cytoplasm, becomes a tiny cell as they are more efficient at locomotion
Not moving yet
Zinc ions and different compositions in the tubular fluid activate it to be mobile
These changes are accomplished without any new gene expression or protein synthesis since spermatids lack transcription and translation machinery
No further change in the nuclear consistency, stays as haploid cell
Change in shape to a more streamlined cell better suited to motility
Acquisition of tail structure
Nuclear compaction: lacks big head, small compacted head
Microvilli become concentrated to one side of the cell, allowing motility capability
DNA is compacted in mammalian sperm, lack large nucleus, easier to maneuver
DNA is normally packaged in histone packaging (large of gaps), all cells are virtually packaged in this way, efficient system as DNA forms histone octamers that form chromosomes
In sperm, histones are taken away and replaced with protamine, creating a more efficient packaging system, chromatin develops doughnut structures
Allows you to gain 1/40 volume with protamine packaging
Sperm nuclear compaction, tight chromatin, very little information can move
Some molecular organizing regions that are necessary to allow the protamine to decondense once fertilization occurs
If packaging is not correct, inheritance errors can occur, not transcriptional.
Epigenetic inheritance of acquired traits through sperm RNAs and sperm RNA modifications
Epimutations induced through affects on the chromosome (X-rays and environmental)
Other form of inheritance: Sperm acquire some traits and RNA’s as they pass through the male reproductive tract through RNAs through extracellular vesicles that contain regulatory RNAs
Occurring in the testis, certain temporal distribution of types of RNAs that during the maturation process in the epididymis which allow some traits to be inherited
May translate to some RNAS being available to help the embryo develop
May be an association with paternal age and perinatal outcomes
More adverse events when father is older regardless of maternal age
Small gestational babies, prematurity
Older fathers may be more prone to fathering children with neurodegenerative disorders, small increases
Some concern as the male ages, sperm is not protected from aging process
Sperm
Specialized membrane that surrounds the head
How mitochondria provides energy for the tail to move, particular structure of tail
Regions of mature spermatozoa
The acrosome (cap): holds digestive enzymes for fertilization, special membrane. Receptors that bind during the process of fertilization
Head (nucleus): stores DNA
Mid-piece (mitochondria): source of ATP
Flagellum (tail): locomotion
Good sperm: nucleus is very dark, condensed, traits of membrane surrounding it, midpiece is slender
Bad sperm: nucleus is gray, patchy. Midpiece is swollen
Loss of cytoplasm: Sertoli cell macrophage property, cross talk with sperm cells
As sperm develops, cytoplasmic droplets aggregates around sperm are sucked into the Sertoli cell
Symbiotic relationship between sertoli cell and developing sperm is important in controlling structure and differentiation of sperm
Particular microtubule pattern: important in allowing sperm to create motility
9+2 microtubule pattern, general structure, allows normal motility
Zona pellucida proteins become very important and allow binding to the egg
During epididymal maturation, stabilization and fertilization proteins that are occurring in the membrane that allow for fertilization, movement, interaction with female reproductive tract, cells surrounding the egg and the zona pellucida
Specific capacitation: modifications in the sperm membrane readying for fertilization that allow for greater motility, one sperm is favored to reach the egg
Cellular, genetic and chromatin changes at the different stages of spermatogenesis and sperm cell maturation are all important in allowing the sperm to gain the characteristics for fertilization and normal development
Translation, transcription, and recombination happen during early stages but are suppressed once the nucleus is heavily compacted
As sperm passes through reproductive tract, acquiring epigenetic characteristics that allow it to give certain traits to the offspring
Delivery of RNAs present in human sperm
Some RNAs appear to be important for the early stages of embryonic development
Early embryo mostly driven by maternal messenger RNA in the oocyte, indication that there are some factors that sperm bring along (paternal RNAs) that influence development
Fertilization
Key event during spermatogenesis and spermiogenesis, want sperm nucleus to decondense and be protaminated
Once fertilized, want protamines to be replaced with histones to have normal histone, nucleated chromosome DNA.
Sperm entry
Male pronuclei decondensation
Sperm enters the oocyte, want protamines to be taken out, histones to be replaced in. Mechanisms within the oocyte strip out protamines, nucleus is changed into a more histone-packed DNA
At same time, male proncelus has to find the female pronucleus. Sends out microvili actin fillaments to search for the female pronuclei, expanding out to draw in the pronuclei
Found pronuclei, both now decondensing and forming pronuclei
Syngamy: 2 haploid cells coming together to make a normal ploidy during fertilization
Imprinting: control on the x and y chromosomes of certain genes
Mostly done by methylation patterns, male pronuclei is very heavily methylated, remains methylated during gametogenesis
Difference in the fertilization and implantation process in both how the male and female gametes are methylated (male sex chromosomes)
Many genes controlled by this mechanism
Imprinting allows for the maintenance of certain genes to ensure that there aren’t traits that are affected by the x chromosome
Sperm
Different species have different lengths of sperm
Human: 65 micrometers, rounded
Mouse: 110 micrometers, most rodent species have a very characteristic beak
Drosophila fruit flies: very long, 58 millimeters
Whale: 56 micrometers
Spermatogenesis
The unique process can result in the production of up to 200 million spermatozoa daily
Very different from conservative activity that controls egg production
WHO: normal semen variables, created by following new couples trying to conceive
Healthy ejaculate
Successful pregnancies: total sperm count in ejaculate between 40 million or more → 928 million.
Ejaculate volume: 1.5-7.6 ml
Sperm concentration, more than 15 million (15-259 million per mL)
Total motility: over 40% is moving (40-81%)
Progressive motility: 32-75% around 40%
Sperm morphology: more than 4% to have normal shape (4-48%)
Not as important compared to motility
Male Infertility: recognized as the single most common cause of infertility with sperm defects accounting for around 30-50% of cases presenting to fertility clinics
Semen analysis: count, motility, shape → assessment
Reference values for male infertility
Count: less than 15 million/ml (Oligozoospermia)
Total motility: less than 40% motile (Asthenozoospermia)
Morphology: less than 15% normal forms (teratozoospermia)
Low numbers + poor motility is oligoasthenozoospermia
Low numbers, poor motility, and poor morphology: oligoasthenoteratozoospermia (OAT)
Azoospermia: obstructive
No sperm in ejaculate
Ejaculatory duct obstruction
Congenital bilateral absence of the vasa deferential → during the fetal development, vas deferens weren’t developed properly
There is a strong association between CBAVD and mutations of the cystic fibrosis transmembrane conductance regular CFTR
Treatment: surgically remove sperm
Azoospermia: non-obstructive
Chromosomal abnormalities resulting in impaired testicular function
Y-chromosome microdeletions leading to isolated spermatogenic impairment
Problem within spermatogenesis itself
Spermatogonia, but not moving forward to make elongated spermatids
Virtually impossible to treat
Certain genes called asospermic factor genes located on y chromosome
AZF A → Sertoli cell only patients
AZF B and C → spermatogenic arrest, indication of nonobstructive
Fertilization: mix sperm and egg together in vitro
Routine IVF: put about 100,000 sperm
Male factor patients, treat male factor infertility by taking single sperm and injecting it into the egg directly,
Intracytoplasmic sperm injection (ICSI), first done in 1992
Pipette, 5-6 microns wide → Oocyte 135 microns wide
Egg membrane (oolemma) is so watery that you cannot tell where the egg is injected
Birth rates equivalent to regular IVF
First IVF pregnancy in 1978
IVF developed to treat women with blocked fallopian tubes
Micromanipulation techniques used to treat infertile men (subzonal insemination) (SUZI) partial zona dissection (PZD)
ICSI: More than 70% of cases worldwide are done using ICSI
Sperm Nuclear Determinants of Reproductive Outcomes
Aneuploidy, y-chromosome microdeletions, epigenetic
Abnormalities in the sperm
Can impact fertilization, embryo development
Pregnancy loss: could be related to sperm
As male ages, the effects on sperm, live pregnancy rate drops
Advancing Paternal Age and Autism
Significant monotonic association between advancing paternal age and the risk of autism
Offspring of men 40 years or older were 5.75x more likely to have ASF compared with offspring of men younger than 40
Increase in DNA damage with age, increasing mutation rate
Strong indications with age
Paternal influence on fertilization, embryo development, pregnancy, fertility, future generations