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PHAR 303 > L20. Reproductive Toxicology > Flashcards

L20. Reproductive Toxicology Flashcards

1
Q

What are the steps of the reproductive cycle?

A
  1. Gamete production and release
  2. Fertilization
  3. Zygote transport
  4. implantation
  5. Embryogenesis
  6. Fetal development
  7. Parturition
  8. Lactation and Postnatal development
  9. Growth and Development
  10. Sexual maturation
    (cycle restarts)
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2
Q

What are the different testing phases of the reproductive cycle for a reproductive toxicant?

A

Segment 1: Testig fertility

  1. Gamete production and release
  2. Fertilization
  3. Zygote transport
  4. implantation

Segment 2: Testing embryotoxicity and teratogenicity

  1. implantation
  2. Embryogenesis
  3. Fetal development
  4. Parturition

Segment 3: pre-postnatal toxicity

  1. Parturition
  2. Lactation and Postnatal development
  3. Growth and Development
  4. Sexual maturation
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3
Q

Explain the key points in female germ cell development.

A
  • Fetal/pre-pubertal phase: Approximately 50 million oocytes are made during fetal life. By birth, the number is reduced to 3 million. At birth there is only one cellular layer around them, they are called primordial follicles and they are arrested in meiosis in the dictyate state until puberty (dormant).
  • Pre-ovulatory phase: At puberty, the influence of LH and FSH leads to the development of the follicle from the primordial to primary meaning there is more than one layer. This is where the follicle starts releasing androgens and estrogens. As the layers increase you get the secondary follicle, then a hole forms in the middle called an antrum.
  • Ovulatory phase: Then a much bigger ovulatory follicle is developed, the egg is released at ovulation and can be fertilized, what is left over is the corpus luteum = yellow body. There are blood vessels in the corpus luteum, and those cells are necessary for releasing hormones during the initial phases of pregnancy if fertilization occurs.
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4
Q

Explain the mechanism of hormonal control in women.

A
  1. The CNS send impulses to the hypothalamus which releases GnRH.
  2. GnRH stimulates the Anterior pituitary to release LH which stimulates the Theca cells, FSH to stimulate the granulosa cells, and prolactin to stimulate the corpus luteum.
  3. The Theca cells produce testosterone in response to LH which also stimulate the granulosa cells to release estrogen. The corpus luteum releases progesterone. These cycle back to feedback on both the hypothalamus and the pituitary. They also go on to affect tissues such as the vagina, the cervix, the uterus, and the oviduct.
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5
Q

Explain ovulation in the menstrual cycle.

A

Estradiol increases as the antrum gets bigger. The increase in estradiol drives the synthesis of LH. When the LH is high it causes the follicle to rupture and the egg to come out. What is left, the corpus luteum, produces progesterone and estradiol for a defined lifespan. If the egg is not fertilized it dies, if it is, it will produce hormones that will allow the maintenance of the endometrial lining.

In the proliferative phase of the endometrium, the cells are under the control of estradiol which allows for cell division. In this phase the cells don’t secrete much but the lining of the endometrium gets thicker. In the secretory phase the progesterone takes over which blocks the receptors for estradiol and allows the cells to become much bigger (stops dividing). In this phase the cells secrete.

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6
Q

What animal is good for studying reproduction compared to humans? What animals are not good?

A

The rat is a good model.
Rabbits and cats are induced ovulators meaning that when they are mounted, it will stimulate the release of a follicle, they don’t have a cycle. Thats why they are rarely used.

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7
Q

What affect do chemotherapeutic agents have on ovarian function? why?

A

Chemotherapeutics cause ovarian dysfunction and interfere with the normal cycle. They affect the active phase of granulosa cell division, so even though the germ cells are not dividing actively, the cells that support oocyte growth are. So drugs that affect cell division will affect female fertility.

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8
Q

What do we analyze when looking for the toxicity of a toxicant on the female reproductive system?

A
  • Body weight
  • Ovary weight
  • Hypothalamus
  • Pituitary
  • Endocrine
  • Oviduct
  • Uterus
  • Cervix
  • Vulva
  • Vagina
  • Fertility
  • IVF (to see if the problem is with the egg itself or with the reproductive system as a whole)
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9
Q

How can we analyze the ovary in order to test the toxicity of a toxicant?

A

You can test the:

  • Organ weight
  • Histology: See if chemicals affect the relative proportion of different follicles
  • Number of oocytes
  • Rate of follicular atresia: The normal fate of a follicle is atresia. For the oocyte to be ovulated it needs to go through all of the right signals.
  • Follicular steroidogenesis
  • Follicular maturation
  • Oocyte maturation
  • Ovulation rate
  • Luteal function (corpus luteum function): The luteal cells (surround the follicle) make androgens in response to LH. The granulosa cells that are right around the oocyte inside the follicle make estrogen in response to FSH. So the Luteal cells make androgens, which cross into the granulosa cells and then is used to make the estrogens in there.
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10
Q

What are the functions of the testes?

A

To produce gametes and hormones.

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11
Q

Where are sperm made? What happens once they are made?

A

Once sperm are made in the testis, they are released but cannot swim or recognize an egg until it passes through the epididymis, then the vas deferens, and secretions from the seminal vesicles and the prostrate come together to form semen, and you have ejaculation.

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12
Q

What is the mechanism of hormonal control in men?

A
  1. The CNS send impulses to the hypothalamus which releases GnRH.
  2. GnRH stimulates the Anterior pituitary to release LH which stimulates the Leydig cells and FSH to stimulate the Sertoli cells.
  3. The leydig cells cells produce testosterone in response to LH and the Sertoli cells release inhibin in response to FSH.
  4. Inhibin is a protein hormone that feedbacks to inhibit the PITUITARY. Cannot feedback to inhibit the hypothalamus because proteins can’t cross the BBB. Testosterone can feedback on both the hypothalamus and the pituitary. Testosterone also goes on to feedback on tissues such as spermatogenesis, accessory sex organs, and masculinization.
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13
Q

Where is the testosterone levels higher? Why?

A

Intra gonadal T is 10-100 times higher than that in the peripheral circulation. Very high local testosterone is needed to promote spermatogenesis.

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14
Q

Where are the seminiferous tubules located? What is its purpose?

A

They are located in the testis and it is where the sperm is made.

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15
Q

Where are the leydig cells located? What do they do? Where are the sertoli cells located? What is their function?

A

Leydig cells are located in the interstitial space (interstitium) of the testes and it is where the steroids are made.
The sertoli cells are located within the seminiferous tubules. They are the “nurse cells” that aid in spermatogenesis.

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16
Q

How is testosterone synthesized? What is its most active version?

A

Acetyl-CoA -> cholesterol -> testosterone

It can be converted into dihydrotestosterone which is its most active version.

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17
Q

What is the duration of spermatogenesis in humans? Rats? mice?

A

Humans: 64 days + 10 days to pass through epididymus
Rats: 48-52 depending on species
Mice: 35 days

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18
Q

How does the duration of spermatogenesis of men affect the detection of toxicants?

A

It takes 64 days for spermatogenesis to occur plus another 10 days for sperm to pass through epididymus. So if you are exposed to a toxicant today, the effect may not be seen for several months afterward. If you stop the exposure, it will take months to go back to normal.

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19
Q

What are the potential target sites for male reproductive toxicants?

A
  1. CNS
  2. Pituitary
  3. Spermatogenesis
  4. Epididymal maturation
  5. Fertilization
  6. Paternal developmental toxicity
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20
Q

What is the cycle of spermatogenesis?

A
  1. Start with a spermatogonia, go through Mitotic and meiotic divisions (called spermatocytes after meiosis 1, spermatid after meiosis 2)
  2. Stop meiosis and start to change cell from round normal appearing cell (haploid)
  3. Sperm concentrates cytoplasm and sheds it
  4. It compacts chromatin to 1/6th of the size of what you would have in a normal nucleus
  5. It forms a tale to swim
  6. Develops a cap (acrosome) to recognize and fertilize an egg (now called spermatozoa - sperm)
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21
Q

What toxicants affect spermatogenesis and how?

A
  1. Vitamin A: Cannot move into meiosis without vitamin A. Stem cells will divide, spermatogonia will be formed (dividing male germ cells), but at some point you have to enter meiosis which needs Vit A. If you lack it, then you block meiosis so you cannot produce sperm.
  2. Zinc: if you have too little zinc, you will not get proteins to bind DNA, too much zinc you will block normal reproductive function.
  3. CCl4 (carbon tetrachloride)
  4. X- Rays: great at killing spermatogonia and spermatogonia division
  5. Cyclophosamide: hit early stages of spermeogenesis
  6. Chlorambucil: affect the spermatids
  7. Acrylamide: affects completed spermatozoa
22
Q

What is the importance of the blood testis barrier? What cells are responsible for this?

A 
Sertoli cells (in the seminiferous tubules) throw out projections to make tight junctions. Those form the blood testis barrier. Once spermatozoa go from the basal compartment to the adluminal compartment, they start making a lot of proteins that are not seen in any other cell in the body therefore making them immunogenic. So if you take a sperm and inject it into the circulation, you will get an immune response. The blood testis barrier (sertoli cells) creates an immune protected area. 
If you isolate sertoli cells and put them around other somatic cells that would normally cause an immune response, it will prevent an immune attack. They secrete a bunch of molecules that suppress the immune system.
23
Q

What drugs are gonadotoxic to men?

A 
Busulfan 
Chlorambucil 
Cyclophosphamide 
Nitrogen mustard 
Methoxy acetic acid 
Dinitrobenzene 
Doxorubicin 
Corticosteroids 
Methotrexate 
Procarbazine 
Vincristine 
Vinblastine
- A lot are Anticancer: If producing 1000 sperm every heart beat = a lot of cell divison
24
Q

What material is toxic to the male reproductive system?

A

Heavy metals such as Cadmium, zinc, lead, chromium, cobalt, platinum (affects germ cells, leydig cells, and sertoli cells), and vanadium.
- Cisplatin is a very common administered drug for testicular cancer, which contains platinum affecting all 3 major cell types.

25
Q

What are the functions of the epididymus?

A
  • Absorption
  • Secretion
  • Transport
  • Maturation (also gain the ability to swim)
  • Storage
  • At time of ejaculation the tail of the epididymus is what contracts to push the sperm through the vas deferens to the outside of the body.
26
Q

According to the WHO in 2010, what are the semen analysis standards?

A 
Volume: 1.4 ml
Total number of sperm: 39 million
Sperm concentration: 16million/mL
Total motility: 42%
Progressive motility:  30%
Vitality:  54%
Sperm morphology:  4%
- If the analysis comes in below these numbers, then there is a fertility problem
27
Q

What happens in men who have cryptorchidism?

A

Meiosis takes place in the scrotum, for it to occur it needs to be 3-4 degrees below core temperature. Over that, meiosis wont happen. Men whose testis are retained in the body (cryptorchidism) are infertile because they can’t go through meiosis.

28
Q

Explain a computer aided semen analysis.

A
  • A motility assessment: put cells in chamber and in this machine that has a microscope camera and computer. Can monitor the path of the sperm, can quantify how long hey move and how efficient they move.
  • Essential in toxicology testing because many chemicals will affect the ability of a sperm to swim. Before it was observed through microscope.
29
Q

Explain the packaging process of sperm chromatin.

A

Somatic cells have nucleosomes and histones. In sperm, it kicks off most of the histones, replaces them by transition proteins, those are kicked off and replaced by protamines that allows for the DNA to be compacted much more tightly. Protamines are very rich in sulfide groups: allows for sulfide links and tight compactions. Because of this, DNA is six-fold more compacted In sperm than in somatic cells.
- Sperm DNA loop domains associated with nuclear matrix at specific sites which assures a very organized structure.

30
Q

How can one assess the quality of sperm?

A

You can assess the quality of sperm through chromatin biomarkers. Chromatin quality can be assessed. Many sperm toxicants do not inhibit the making of a sperm but they damage chromatin = consequences.

You can assess the:
Sperm decondensation rate
Breaks and cross-links and integrity of chromatin Chromatin template function
Chromatin structure
Chromatin epigenome (Methylation, histones that remain, non coding RNA)

31
Q

What methods can be used to assess sperm quality by looking at chromatin biomarkers?

A
  1. Comet assay: take sperm nuclei, decondense them, put them in an electric field, and see whether some of the DNA moves, the more DNA moves the more damage there is (more bits and pieces broken up)
  2. Tunel: assessing if there are breaks in DNA, shows how many free prime 3 hydroxy open ends you have
32
Q

What do we analyze when looking for the toxicity of a toxicant on the male reproductive system?

A
  • Testis
  • Epididymis
  • Accessory Glands: Prostate, Seminal Vesicles, Cowper’s glands
  • Semen
  • Endocrine
  • Sperm motility
  • Sperm chromatin integrity
  • Fertility
  • In vitro fertilization
33
Q

How can the Testis be tested in order to assess a reproductive toxicant?

A 
Size in situ
Weight
Spermatid reserves
Gross and histological evaluation 
% nonfunctional tubules
% tubules with sperm in lumen 
Tubule diameter
Count of leptotene spematocytes 
Number and size of Leydig cells 
Testosterone production
34
Q

Explain how a toxicant can affect the histology of the rat testis.

A
  • Just by doing cross sections and looking at tissues you can tell how the toxicant is working.
  • In a rat treated by methoxy acetic acid for 24 hours, the testis was disorganized
  • In a rat treated by m-dinitrobenzene for 24 hours, there were vacuoles and abnormalities in the testis
35
Q

What are the standard reproductive endpoints? What are the limitations and advantages of each?

A
  1. Fertility
    - Limitations: Insensitive. You can suppress spermatogenesis in rat testis by 95% before you start seeing effects on fertility due to the excess number of sperm.
    - Advantage: Integrates all reproductive functions
  2. Testicular histology
    - Limitations: Subjective, not quantitative
    - Advantage: Information on target cells
  3. Testis weight:
    - Limitations: Less sensitive than sperm count, affected by edema
    - Advantages: Rapid, quantitative
    - 90% of the weight of a testis is germ cells. Having a 10% decrease is significant.
  4. Gene expression
    - Limitations: “overly” sensitive so it’s hard to assess what matters. (may not have physiological consequences)
    - Advantages: comprehensive
36
Q

Over the past 50 years what has been happening to sperm concentrations?

A

It has been declining.

37
Q

What is the trend in testicular cancer over the years?

A
  • Rate of testicular cancer is increasing.

- Treatment has improved and rate of death gone down. But the drugs used usually cause infertility.

38
Q

What is hypospadius and what is the trend?

A

The opening of the urethra is not at the tip of the penis in many boys but rather the opening is along the shaft of the penis. The incidence of hypospadius is increasing.

39
Q

What is TDS? How is it caused? What is its mechanism?

A
  • Testicular dysgenesis syndrome
  • Testicular dysgenesis can be caused by genetic defects (minor) and/or environmental factors and endocrine disrupting chemicals (major).
  • TDS leads to disturbed sertoli cell function which causes a decrease in germ cell differentiation. This leads to decreased sperm quality and carcinoma in situ (testicular cancer)
  • TDS also leads to a decrease in leydig cell function which causes a decrease in androgens and a decrease in insl3. A decrease in androgens causes Hypospadias and testicular maldescent. And insl3 (which drives testicular descent from peritoneal cavity to the scrotum. The incidence of cryptorchidism has also been increasing) causes testicular maldescent as well.
40
Q

What is cyclophosphamide?

A
  • CPA
  • Very commonly used immunosuppressant/anti cancer drug.
  • Used for Hodgkin lymphoma. Young kids between 3-12 who have lymphoma will all be treated with CPA.
  • It is a bifunctional alkylating agent affecting DNA, RNA, and proteins.
41
Q

What is the timing of the effects of cyclophosphamide on spermatogenesis?

A

Rats treated for 9 weeks.

  • After 1 week, the effect has to be on the sperm residing in the epididymus.
  • If see an affect between 2-4 weeks, then the effect has to be on spermatids (haploid cells that are reshaping themselves).
  • If the effect shows in 5-6 weeks, its an effect on the spermatocytes (meiosis).
  • If 7-9 weeks it’s on spermatogonia (the stem cells or on the Mitotic phase).
  • Remember how long it takes to go through a full cycle of spermatogenesis. If the affect is seen quickly, it means it has to be affecting the later stages of spermatogenesis first, because you are seeing the effects on spermatogenesis quickly, and so on.
42
Q

What are 2 tests that can be used to assess the breaks and cross-links and integrity of chromatin when using chromatin biomarkers to assess sperm quality? Explain.

A
  • FISH aneuploidy assay: Fluorescence in situ hybridization: antibody recognizing part of a chromosome. So you can see which chromosomes are being affected.
  • Comet assay: detects DNA damage through looking at comet tail presence/size.
43
Q

What are the effects of chronic CPA treatment on male rats treated for 9 weeks?

A
  • The biggest effect was on post implantation. There are minor effects at 1 week (epididymus), but the effects are primarily on the spermatids (2-4 weeks).
  • Skeletal abnormalities in offspring (only father is exposed - sperm damaged. Female is totally drug-naive)
  • Assessing sperm morphology via EM assessment: you can see that it affects the formation of the sperm as the tail is being made.
  • Assessing sperm quality through chromatin biomarkers: Breaks and cross-links and integrity of chromatin. Using A FISH aneuploidy assay: the overall effect is the doubling of abnormal chromosomes.
    Using a comet assay: there was much more movement (bigger comet tail) in the cyclophosphamide treated group than in the control group.
44
Q

What is the overall effect of CPA on male rats?

A
  • Leads to abnormal progeny in the spermatogonia
  • Leads to pre-implantation loss in the spermatocytes
  • Leads to post-implantation loss in spermatids and spermatozoa.
  • Can also affect early embryos
45
Q

On what areas does paternal CPA exposure affect early embryos development?

A 
- The messed up sperm from the testis can now fertilize an egg. What happens to zygote?
• Chromatin remodelling
• Pronuclear formation
• Zygotic genome activation 
• Cell lineage determination
46
Q

Explain CPAs effect on the pronuclear stages in zygotic development.

A
  • Normally, when a sperm enters and egg the male pronucleus is the bigger one, and during the phases of the zygote the pronuclei (maternal and paternal) move together and eventually merge.
  • When the male is treated with CPA, the male pronucleus is getting bigger faster.
  • Even though the females were not exposed to CPA, if they were mated with the CPA exposed males, the female’s pronuclei sensed the males abnormal pronuclei and also started expanding faster than normal.
47
Q

What affect does CPA have on the epigenetic marks in the zygote?

A
  • Normally, histone Acetylation and deacetylation regulate gene transcription. Core histone acetylation is correlated with activation of the zygotic genome in early embryonic development.
  • CPA male pronucleus: have a lot more histone acetylation occurring in early phases.
  • Female pronucleus (of the females mated with CPA males, but the females are still CPA naive) have more histones acetylation than in control. So the epigenetic mark in the female is also responding to the damages to the male pronucleus. (Again only the male is being treated with CPA)
48
Q

What explains the reaction of the female pronucleus to the males CPA treated pronucleus?

A

A damaged sperm will be sensed in the zygote by mechanisms that the female oocyte has. We don’t understand how they communicate with each other. There is a program in the female that repairs some of the male damage, and the extent of the repair is variable.

49
Q

How did they assess if the affects of CPA were multigenerational?

A

F0 male: seen the drug. Mate with drug naive females
F1: not seen drug + mated with others whose dad has seen drug
F2: effects? If male is affected: transgenerational
If affect male in F1: multigeneration
For female you have to get to F3

50
Q

What were the generational effects of paternal CPA exposure?

A
  • Pre-implantation loss: no change
  • Post implantation loss: increase
  • Malformations: increase
  • Paternal CPA affects the F2 generation -> transgenerational
51
Q

What is Vinclozolin? What test was done to see the gremline transmission?

A
  • Used to be spread on golf curses to make nice grass

- Treated males, looked at F1, mated F1, looked at F2 mated again, looked at F3

52
Q

What are the results of the Vinclozolin test? Why is this important?

A
  • The proportion of apoptotic cells at a different stage in spermatogenesis is higher in F1, but stays similarly High through 2,3,4.
  • This is important for toxicology because usually studies are done on 2 generations. Doing studies on 3-4 generations is expensive especially with trying to stop using animals. So how will we study this effect?

PHAR 303 (21 decks)

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