59th Annual Meeting & ToxExpo March 15–19, 2020 • Anaheim, California Continuing Education Course Sunday, March 15 | 1:15 PM to 5:00 PM
PM14: The Male Reproductive Tract: Development, Toxicology, and Pathology
Chair(s) Vicki Sutherland, NIEHS/NTP Nicole Principato, Bristol-Myers Squibb Company
Primary Endorser Reproductive and Developmental Toxicology Specialty Section
Other Endorser(s) Comparative Toxicology, Pathology, and Veterinary Specialty Section Regulatory and Safety Evaluation Specialty Section
Presenters Justin Vidal, Charles River Kim Boekelheide, Brown University Catherine Picut, Charles River Cynthia Willson, Integrated Laboratory Systems Inc. Course Participant Agreement
As a course participant, you agree that the content of this course book, in print or electronic format, may not, by any act or neglect on your part, in whole or in part, be reproduced, copied, disseminated, or otherwise utilized, in any form or manner or by any means, except for the user’s individual, personal reference, or in compliance with the US Government Copyright Law as it pertains to Fair Use, https://www.copyright.gov/fair-use/more-info.html.
The author(s) of each presentation appearing in this publication is/are solely responsible for the content thereof; the publication of a presentation shall not constitute or be deemed to constitute any representation by the Society of Toxicology or its boards that the data presented therein are correct or are sufficient to support conclusions reached or that the experiment design or methodology is adequate.
Continuing Education Committee Udayan M. Apte, Chair Cheryl E. Rockwell, Co-Chair
LaRonda Lynn Morford Alexander Suvorov Dahea You Member Member Postdoctoral Representative William Proctor Lili Tang Lisa Kobos Member Member Student Representative Julia Elizabeth Rager Terry R. Van Vleet Cynthia V. Rider Member Member Council Contact Jennifer L. Rayner Kevin Merritt Member Sta Liaison
11190 Sunrise Valley Drive, Suite 300, Reston, VA 20191 Tel: 703.438.3115 | Fax: 703.438.3113 Email: [email protected] | Website: www.toxicology.org
#2020SOT 2 #toxexpo The Male Reproductive Tract: Development, Toxicology, and Pathology
1:30 PM–2:15 PM An Overview of the Male Reproductive System: Applied Anatomy and Physiology from a Pathologist’s Perspective Justin Vidal, Charles River, Mattawan, MI 4
2:15 PM–3:00 PM Prenatal Development of the Male Reproductive Tract in the Rat, Dog, and Human: Critical Developmental Windows and Later-Life Consequences of Exposure Kim Boekelheide, Brown University, Providence, RI 34
3:00 PM–3:30 PM Break
3:30 PM–4:15 PM Postnatal Development of the Juvenile Male Reproductive Tract in Rats: Microscopic Evaluation, Interpretation, and Time Points of Toxicologic Significance Catherine Picut, Charles River, Durham, NC 61
4:15 PM–5:00 PM Pathology in Reproductive Toxicology Assessments and the Role of Stage-Awareness for Testis Evaluation Cynthia Willson, Integrated Laboratory Systems Inc., Research Triangle Park, NC 90
#2020SOT 3 #toxexpo Presenter 1 | PM14
An Overview of the Male Reproductive System: Applied Anatomy and Physiology from a Pathologist’s Perspective
Justin Vidal, DVM, PhD, DACVP Charles River Mattawan, MI Phone: 269.668.3336 ext. 1071 Email: [email protected]
1
Conflict of Interest Statement
The author declares no conflict of interest.
2
#2020SOT 4 #toxexpo Presenter 1 | PM14
reviatio s
• DHT: dihydrotestosterone • OAT: organic anion transporters • E2: estradiol • OATP: organic anion transporting • FSH: follicle stimulating hormone polypeptides • GnRH: gonadotropin releasing • OCT: organic cation transporters hormone • OECD: Organisation for Economic • ICH: International Council for Co-operation and Development Harmonisation of Technical • T: testosterone Requirements for Pharmaceuticals • US FDA: US Food and Drug for Human Use Administration • IHC: immunohistochemistry • LH: luteinizing hormone
3
tro ctio
Before administering the first dose in healthy male volunteers, the sum total of the assessment on the male reproductive system falls on the study (and peer-review) pathologist(s)!
4
#2020SOT 5 #toxexpo Presenter 1 | PM14
i a ce
• ICH: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5(R2) • Note 12 (4.1.1) • Information on potential effects on spermatogenesis can be derived from repeated-dose toxicity studies • Histopathology of the testis has been shown to be the most sensitive method for the detection of effects on spermatogenesis
5
i a ce o ti e
• ICH: Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals M3(R2) • 11.1 Men • Men can be included in Phase I and II trials before the conduct of the male fertility study since an evaluation of the male reproductive organs is performed in the repeated-dose toxicity studies (Note 2) • A male fertility study should be completed before the initiation of large scale or long duration clinical trials (e.g., Phase III trials) • Note 2: An assessment of male and female fertility by thorough standard histopathological examination on the testis and ovary in a repeated-dose toxicity study (generally rodent) of at least 2-week duration is considered to be as sensitive as fertility studies in detecting toxic effects on male and female reproductive organs
6
#2020SOT 6 #toxexpo Presenter 1 | PM14
i a ce o ti e
• ICH: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals S6(R1) • 5.2 Fertility • It is recognized that mating studies are not practical for nonhuman primates (NHP). o ever he the P is the o l releva t species the pote tial or e ects o ale a e ale ertilit ca e assesse eval atio o the repro ctive tract orga eights a histopatholog eval atio i repeat ose to icit st ies o at least o ths ratio si g se all at re Ps
7
• est o (Repro./Dev. Tox. Screening Test) and (Combo. Repeated Dose Tox. Study with the Repro./Dev. Tox. Screening Test): • “Detailed histological examination should be performed on the ovaries, testes, and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure)” • est o (Two-Generation Reproduction Toxicity): • “Detailed testicular histopathological examination . . . should be conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells into the lumen” • i a ce : Guidance Document for Histologic Evaluation and Reproductive Tests in Rodents
8
#2020SOT 7 #toxexpo Presenter 1 | PM14
P
• ealth ffects est uidelines . eproduction and ertility ffects • “. . . testicular histopathological examination should be conducted in order to identify treatment related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells into the lumen”
ac gro
• ge • uberty • P • pecies differences • lso consider strain and supplier differences • athology is often lin ed to fertility infertility and not necessarily the overall health of the animal • Definition of adversity can be challenging
#2020SOT 8 #toxexpo Presenter 1 | PM14
ale epro ctive ste — poi ts i a epeat ose o t
rga eight istopatholog estis pidid mis rostate eminal esicles ituitar ammar gland rats onl
asic ato
pi i is
ere t cts Pa pi i or ple s
as e ere s
estis
#2020SOT 9 #toxexpo Presenter 1 | PM14
• ete is peripherall located at the proximal pole arro s • pposite the large essels arro heads • o lobular pattern ith minimal connecti e tissue
• • erm cells • ertoli cells • asement membrane • eritubular m oid cells
• • e dig cells • acrophages • asculature
#2020SOT 10 #toxexpo Presenter 1 | PM14
tage – tage – tage
Β catenin emonstrates the location and d namic nature o the blood testis barrier. ote that the barrier is ithin the tubule
#2020SOT 11 #toxexpo Presenter 1 | PM14
Toxicokinetics: systemic exposure of compound “X” on Days 1 and 7
#2020SOT 12 #toxexpo Presenter 1 | PM14
1 our
o e ody autoradio ram: in e ra dministration of [14C] Labeled Compound “X” at 300 mg/kg
1
ours
24 hours
o e ody utoradio ram: in e ra dministration of [14C] Labeled Compound “X” at 300 mg/kg
#2020SOT 13 #toxexpo Presenter 1 | PM14
3 a
a
ole bod uto ad og am ngle al dm n t at on o [14C] Labeled Compound “X” at 300 mg/kg
1
odel ng t e p ed ted te t on ent at on du ng 400 epeat do ng ba ed on 3 0 t ue d t but on tud e Compound “X” 300 for compound “X” and compound “Y.” Compound 0 “Y” has the same target 00 engagement but d e ent
1 0 em al la ote t e Compound “Y” 100 d e en e n t me to t ue tead tate and t e 0 d e en e n t ue 0 lea an e 0 30 0 0 1 0 1 0 1 0
#2020SOT 14 #toxexpo Presenter 1 | PM14
p Comp on
p um o C o u on o p d d m n p m o n n m 14 1 . 1. ou 1 . 34. o 13. 4.4 10 . 40 10 1 10. 4 .4 11 um n 1 4 mo 10 3
3
o n on –
p on p p o n p m o Con o –
ote nh b t on of spermatogon a m tot c act t leads to loss of spermatogon a and ult matel loss of more mature cell t pes. etect ng the n t al loss of spermatogon a can be d ff cult and loss of preleptotene spermatoc tes earl spermatoc tes s often the f rst read l dent f able f nd ng.
4
#2020SOT 15 #toxexpo Presenter 1 | PM14
elongated spermat ds
round spermat ds
pach tene spermatoc tes
preleptotene spermatoc tes
–
elongated spermat ds
round spermat ds
pach tene spermatoc tes
preleptotene spermatoc tes
#2020SOT 16 #toxexpo Presenter 1 | PM14
Changes can be obser ed th n the ep d d m s n appro matel eeks
#2020SOT 17 #toxexpo Presenter 1 | PM14
emember spermatogon a are depleted too ust d ff cult to dent f n standard sl des
egenerat on of elongated spermat ds
elongated spermat ds
round spermat ds pach tene spermatoc tes
30
#2020SOT 18 #toxexpo Presenter 1 | PM14
31
• nderstand ng the cell t pe s affected th n the test s s cr t cal to des gn of follo up stud es and s needed before d scuss ng length of dos ng length of reco er endpo nts etc. • h s s ke component of the ha ard character at on and r sk assessment
3
#2020SOT 19 #toxexpo Presenter 1 | PM14
33
fferent ducts
ete test s
em n ferous tubules
34
#2020SOT 20 #toxexpo Presenter 1 | PM14
fferent fferent ducts ducts
ete ete test s p d d m s test s p d d m s
ats m ce ost mammals although number of tubules and e act po nts of nsert on ar
3
n rats the efferent ducts are embedded n a fat pad h ch s d scarded n most to c t stud es. f a potent al target organ must re uest prospect el
3
#2020SOT 21 #toxexpo Presenter 1 | PM14
est s p d d m s perm are concentrated n efferent ducts as flu d s resorbed
3
perm ranuloma
3
#2020SOT 22 #toxexpo Presenter 1 | PM14
ght ormal Left n lateral d lat on of the sem n ferous tubules th d lat on of the rete test s arro s
3
gher magn f cat on n lateral d lat on of the sem n ferous tubules th d lat on of the rete test s arro s 40
#2020SOT 23 #toxexpo Presenter 1 | PM14
mm
fferent ducts ete test s
41
perm ranuloma
4
#2020SOT 24 #toxexpo Presenter 1 | PM14
p d d m s
fferent ducts amp n form ple us
as deferens
est s
43
e n
rter
44
#2020SOT 25 #toxexpo Presenter 1 | PM14
ascular necros s and nflammat on th tubular degenerat on
4
p d d m s
fferent ducts amp n form ple us
as deferens
est s
4
#2020SOT 26 #toxexpo Presenter 1 | PM14
ead n t al egment
n t al ead egment
od
od a l
a l
4
p thel al acuolat on ncreased lum nal cell debr s
4
#2020SOT 27 #toxexpo Presenter 1 | PM14
aca ues ha e cran al and caudal lobes of the prostate and the cran al lobe ser es a coagulat ng gland l ke funct on
4
orsal
Lateral
entral
0
#2020SOT 28 #toxexpo Presenter 1 | PM14
og rostate Caudal rostate
rostate enc rcles the urethra rostate does not enc rcle the urethra th r ght and left lobes and ha e cran al and caudal lobes o sem nal es cles Large sem nal es cles
1
troph ecreased androgen product on
strogen c st mulat on
Control uamous metaplas a and stromal edema
#2020SOT 29 #toxexpo Presenter 1 | PM14
• pothalamus • onadotrop n releas ng hormone n • tu tar • oll cle st mulat ng hormone • Lute n ng hormone L • rolact n • est s • estosterone • nh b ns • strad ol spec es dependent • er pheral con ers on • h drotestosterone • strad ol
3
aromat ed to
n
egat e feedback nh b ns L
ertol Le d g cells cells estosterone
upports spermatogenes s nabol c effects ccessor se organs local con ers on to
4
#2020SOT 30 #toxexpo Presenter 1 | PM14
• ats • o clear d urnal rh thm • ghl ar able o er the da • peaks do not al a s follo L peaks • ogs • o clear d urnal rh thm • peaks follo L peaks e er hour to 1. hours • gs • o clear d urnal rh thm • peaks follo L peaks e er –3 hrs age dependent • • eaks at n ght • peaks follo L peaks
• atholog assessment pla s a ma or role n understand ng male reproduct e effects • emember spec es spec f c d fferences n anatom and ph s olog • ust ha e clear understand ng of the cell t pes affected n the test s • Carefull cons der t m ng of sample collect on and assessment
#2020SOT 31 #toxexpo Presenter 1 | PM14
• Birth Defects Res B Dev Reprod Toxicol. • Toxicol Pathol • Physiol Rev • Toxicol Pathol • Toxicol Pathol • • • S6(R1) • • Ann N.Y Acad Sci
• Reprod Toxicol • Test No. 416: Two-Generation Reproduction Toxicity • • Test No. 421: Reproduction/Developmental Toxicity Screening Test • Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test •
#2020SOT 32 #toxexpo Presenter 1 | PM14
• Spermatogenesis • •
#2020SOT 33 #toxexpo Presenter 2 | PM14
Prenatal Development of the Male Reproductive Tract in the Rat, Dog, and Human: Critical Developmental Windows and Later-Life Consequences of Exposure
Kim Boekelheide, MD, PhD Brown University Providence, RI Email: [email protected]
1
Disclosure
Current funding from NIEHS. Occasional expert consultant for chemical and pharmaceutical companies, including Tb Alliance and Lantmännen Medical.
2
#2020SOT 34 #toxexpo 34 Presenter 2 | PM14
Bipotential Fetal Testis Development Gonad
Sry
Sox9 • A sequence of molecular and Fgf9 hormonal signaling events drive Wnt4 Cell Migration and Differentiation the bipotential gonad down the Coelomic Mesonephric Mesenchymal path of male differentiation Epithelia Epithelia • Disruption of these signals results in dysgenesis, including intersex Sertoli Cells Myoid Cells Leydig Cells conditions, testicular dysgenesis, SF1 SF1
and abnormalities in the external INSL3 Testosterone Wolffian duct AMH + GCs remodeling genitalia
testis DHT Müllerian duct Cords descent seminal vesicles regression vas deferens epididymis penis and prostate growth
3
Fetal Testis Development
• The order of developmental events is similar across mammals • But the timing, relative to parturition, differs significantly
Human
4
#2020SOT 35 #toxexpo 35 Presenter 2 | PM14
Fetal Male Reproductive Developmental Milestones
Human Genital tubercle forms
External genitalia form Dog Onset HPG axis activity
Rat Plasma androgen levels
0 20 40 60 80 Birth Percentage of fetal life
5
Normal Human Testicular Descent
• Insulin-like 3 protein (Insl-3) is made by Leydig cells and regulates growth and differentiation of gubernaculum, mediating intra-abdominal testicular descent • Androgens produced by the Leydig cell mediate all phases of testicular descent
ENDOTEXT: Hutson et al., INSL3 Cryptorchidism and Hypospadias https://www.ncbi.nlm.nih.gov/books/NBK279106/ androgen
6
#2020SOT 36 #toxexpo 36 Presenter 2 | PM14
Descended testis
Cryptorchid testis
7
ENDOTEXT: Hutson et al., Cryptorchidism and Hypospadias Human Hypospadias https://www.ncbi.nlm.nih.gov/books/NBK279106/
Hypospadias is associated with: • Failure of the urethral meatus to be located on the tip of the glans • Failed ventral fusion of the prepuce, causing a “dorsal hood” • Inadequate growth of the ventral shaft around the urethra, leading to bend
8
#2020SOT 37 #toxexpo 37 Presenter 2 | PM14
Flutamide-Induced Hypospadias in Rat
• lu a ide is a po en androgen recep or an agonis ha produces h pospadias • en ral prepu ial cle is presen in all lu a ide rea ed ales as ell as a pseudo aginal pouch • he inse in sho s he ip o he penis, hich nor all ould no e isi le
Flutamide-Induced Hypospadias in Rat
• he si e o he prepu ial ea us is de ined in red, i h he lu a ide rea ed ale ha ing he longes prepu ial ea us groo e or ales
#2020SOT 38 #toxexpo 38 Presenter 2 | PM14
Anogenital Distance
nogeni al dis ance is a se uall di orphic endpoin use ul in o h ani al and hu an s udies o in utero ale reproduc i e rac de elop en he ac son a ora or , eproduc i e iolog o ice e inar, ugus ,
“We argue that the increasing incidence of reproductive abnormalities in the human male may be related o increased oes rogen e posure in utero, and identify mechanisms by which this exposure could occur.”
e elop en o he ale reproduc i e rac or onal con rol o he e al es is
s rogen suppresses e dig cell es os erone s rogen suppresses a is • nhi i s es is descen • nhi i s er oli cell unc ion • nhi i s asculini a ion • educes sper produc ion
Testis “descends” er oli cell proli era ion in o scro u and ger cell suppor
#2020SOT 39 #toxexpo 39 Presenter 2 | PM14
Diethylstilbestrol (DES)
• owerful synthetic estrogen • rescribed to pregnant women – • trong evidence of effects on male fetuses in animal studies • pidemiologic evidence of reproductive effects in males exposed in utero relatively low ris • uman fetal testis xenotransplants
DES—Animal Studies
Lesions in the reproductive tract of male mice exposed prenatally to DES. Males were the 9- to 10-month-old offspring of CD-1 mice treated with DES (100 μg/kg, subcutaneously) on days 9 to 16 of gestation. Location of lesions Prenatal Incidence of treatment lesions* Testis † Epididymal Accessory sex cysts gland § orn oil
† cryptorchidism and or fibrosis calcification § nodular masses with s uamous metaplasia
#2020SOT 40 #toxexpo 40 Presenter 2 | PM14
• • • • Therefore, ↓Sertoli cells means ↓germ cells and ↓epididymal sperm
—
– – – – –
#2020SOT 41 #toxexpo 41 Presenter 2 | PM14
“While it is clear that hypospadias, cryptorchidism, and contraceptives during pregnancy.”
#2020SOT 42 #toxexpo 42 Presenter 2 | PM14
—
et al.,
ote the a undant estrogen receptor alpha rat α staining in the rat eydig cell compared ith the human, and the corresponding di erence in diethylstil esterol e posure on testis enotransplant production human o testosterone
mmunohistochemistry or α human rat
pidemiological research suggests that male reproductive disorders—including cryptorchidism, hypospadias, in ertility, and testicular cancer—have een increasing in incidence
estis germ cell cancer incidence
#2020SOT 43 #toxexpo 43 Presenter 2 | PM14
Altered Leydig cell function Altered Sertoli cell function
Cryptorchidism Hypospadias Sperm quality/quantity Testicular cancer
Altered Leydig cell function Altered Sertoli cell function
Cryptorchidism Hypospadias Sperm quality/quantity Testicular cancer
#2020SOT 44 #toxexpo 44 Presenter 2 | PM14
Altered Leydig cell function Altered Sertoli cell function
Cryptorchidism Hypospadias Sperm quality/quantity Testicular cancer
Altered Leydig cell function Altered Sertoli cell function
Cryptorchidism Hypospadias Sperm quality/quantity Testicular cancer
support germ cells produce testosterone
become sperm
#2020SOT 45 #toxexpo 45 Presenter 2 | PM14
Altered Leydig cell function Altered Sertoli cell function
Cryptorchidism Hypospadias Sperm quality/quantity Testicular cancer
Hum Reprod
• • ➢ ➢ • “Drug like” mode of action
#2020SOT 46 #toxexpo 46 Presenter 2 | PM14
◼ aternal e o ure → fetal e o ure ◼ ro lacental arrier ◼ eta olite detecti le in urine and amniotic fluid
◼ riticall ill neonate ◼ nten i e medical inter ention ◼ – mg da off et al ◼ “Serious Concern” ◼ Children aren’t small adults . . . ◼ ig er od urden ◼ cti e de elo ment ◼ mmature deto ification function
♂ ♂ ♂ ♀ ♀ ♀
#2020SOT 47 #toxexpo 47 Presenter 2 | PM14
hthalate
♂ ♂ ♂ ♀ ♀ ♀
emini er us C rd e ts YES C rd diameter
hthalate
♂ ♂ ♂ ♀ ♀ ♀
emini er us C rd e ts YES C rd diameter
e di Cell e ts YES Ch lester l s adias Cr t r hidism n enital distan e e di ell h er lasia i le are la retenti n
est ster ne
#2020SOT 48 #toxexpo 48 Presenter 2 | PM14
emini er us C rd e ts YES C rd diameter
e di Cell e ts YES s adias Cr t r hidism n enital distan e e di ell h er lasia i le are la retenti n
emini er us C rd e ts YES emini er us C rd e ts YES C rd diameter C rd diameter
e di Cell e ts YES s adias Cr t r hidism n enital distan e e di ell h er lasia i le are la retenti n
#2020SOT 49 #toxexpo 49 Presenter 2 | PM14
emini er us C rd e ts YES emini er us C rd e ts YES C rd diameter C rd diameter
e di Cell e ts YES e di Cell e ts NO s adias Cr t r hidism h s adias n enital distan e r t r hidism e di ell h er lasia han e an enital distan e i le are la retenti n e di ell h er lasia
emini er us C rd e ts YES emini er us C rd e ts YES emini er us C rd e ts C rd diameter C rd diameter
e di Cell e ts YES e di Cell e ts NO s adias Cr t r hidism h s adias n enital distan e r t r hidism e di ell h er lasia han e an enital distan e i le are la retenti n e di ell h er lasia
#2020SOT 50 #toxexpo 50 Presenter 2 | PM14
emini er us C rd e ts YES emini er us C rd e ts YES emini er us C rd e ts C rd diameter C rd diameter
e di Cell e ts YES e di Cell e ts NO e di Cell e ts s adias Cr t r hidism h s adias n enital distan e r t r hidism e di ell h er lasia han e an enital distan e i le are la retenti n e di ell h er lasia
r m se e endent lterati ns in ene ressi n and est ster ne nthesis in the etal estes ale ats sed t i n ut l hthalate o ico Sci. – . d i . t s i h o ico Sci o ico o ic Sciences l. n . iet i l all ri hts reser ed.
#2020SOT 51 #toxexpo 51 Presenter 2 | PM14
n iron e ers ec –
• •
io o o e ro uc ion –
#2020SOT 52 #toxexpo 52 Presenter 2 | PM14
o ico Sci
Control DBP (500 120 mg/kg) 100 * 80 AGD (%AGD Control)
60 Rat Mouse (GD16–20 Exposure) (GD14–18 Exposure)
#2020SOT 53 #toxexpo 53 Presenter 2 | PM14
↓ ↑ ↔ ↓ ↑ ↔
o ico o ic Sciences –
– o ico o ic Sciences –
#2020SOT 54 #toxexpo 54 Presenter 2 | PM14
140 3 120
100 2 80
60 Serum Serum 1 40
Testosterone (ng/dL) Testosterone 20 Progesterone (ng/mL) Progesterone 0 0 vehicle abiraterone vehicle abiraterone o ico o ic Sciences –
#2020SOT 55 #toxexpo 55 Presenter 2 | PM14
100 40 160 140 80 30 120 60 100 20 80 40 60 20 10 LABC (mg) 40 20 Anterior Prostate (mg) Seminal Vesicles (mg) 0 0 0 vehicle abiraterone vehicle abiraterone vehicle abiraterone o ico o ic Sciences –
140 4
120 100 3 80 2 60 Serum Serum 40 1 20 Testosterone (ng/dL) Testosterone Progesterone (ng/mL) Progesterone 0 0 vehicle DBP vehicle DBP • o ico o ic Sciences –
#2020SOT 56 #toxexpo 56 Presenter 2 | PM14
140 160 50 120 140 100 40 120 100 80 30 80 60 20 60 40 LABC (mg) 40 10 20 20 Anterior Prostate (mg) Seminal Vesicles (mg) 0 0 0 vehicle DBP vehicle DBP vehicle DBP
• o ico o ic Sciences –
4 700
2 600 3 500 400 2 300 200 1 Area Cord
Germ Cells 100 Germ Cells/mm
% Multinucleated 0 0 vehicle DBP vehicle DBP
• •
o ico o ic Sciences –
#2020SOT 57 #toxexpo 57 Presenter 2 | PM14
ad e et al., Env Int’l –
“Overall, despite some inconsistencies across phthalates in the specific outcomes associated with exposure, these results support that phthalate exposure at levels seen in human populations ma have male reproductive effects, particularl and . he relative strength of the evidence reflects differing levels of toxicit as well as differences in the range of exposures studied and the number of available studies.”
• hile the male reproductive tract development se uence is similar across mammals, the timing varies • ale reproductive tract development is hormonall sensitive • oth testis descent and formation of the urethra are androgen dependent • strogenic exposures, such as to dieth lstilbesterol , alter male reproductive tract development • odents ma be more sensitive than humans to estrogenic exposures • esticular sgenesis ndrome is a h pothesis to explain the increased incidence in multiple male reproductive tract developmental abnormalities • evelopmental phthalate exposure in rodents recapitulates several abnormalities • he hthalate ndrome • he developing male reproductive tract sensitivit to phthalate varies across species • at ouse uman
#2020SOT 58 #toxexpo 58 Presenter 2 | PM14
• ielden , algren , ong , taub , ohnson , hou , acharews i . estational and actational xposure of ale ice to ieth lstilbestrol auses ong erm ffects on the estis, perm ertili ing bilit In t and esticular ene xpression. En n l . – . • aido , ensle , iu , allace , orghoff , ohnson , all , oe elheide . etal ouse hthalate xposure hows hat onoc te ultinucleation s ot ssociated with ecreased esticular estosterone. l – . • an e , ies , dwards . he urveillance, pidemiolog , and nd esults rogram ational esource. n E l v . • eger , all , androf , et al., uman etal estis enografts re esistant to hthalate nduced ndocrine isruption. Env n lt t. – . doi . ehp. . • utson . r ptorchidism and pospadias. ul . n eingold , nawalt , o ce , et al., editors. En t t nternet . outh artmouth ext.com, nc. . vailable from https www.ncbi.nlm.nih.gov boo s • ohnson , eger , oe elheide . Of ice and en and ats hthalate nduced etal estis ndocrine isruption s pecies ependent. l . – . doi . toxsci fs . • ehmann , hillips , ar , oster , aido . ose ependent lterations in ene xpression and estosterone nthesis in the etal estes of ale ats xposed to i n but l hthalate. l – .
• artin O , hialis , ester , crimshaw , oobis , oulvoulis . esticular sgenesis ndrome and the strogen pothesis uantitative eta anal sis. Env n lt t. – . doi . ehp. . • c achlan , ewbold , and ulloc eproductive ract esions in ale ice xposed renatall to ieth lstilbestrol. n – . • itchell , harpe , nderson , et al., ieth lstilboestrol xposure oes ot educe estosterone roduction in uman etal estis enografts. n . e . ublished pr . doi . ournal.pone. . • almer , erbst , oller , et al., rogenital bnormalities in en xposed to ieth lstilbestrol In t ohort tud . Env n lt . . ublished ug . doi . . • ad e . ., raun . ., ee er . ., ooper . . hthalate xposure and ale eproductive Outcomes stematic eview of uman pidemiological vidence. Env n Int – . doi . .envint. . . . • harpe , a ebae . re Oestrogens nvolved in alling perm ounts and isorders of the ale eproductive ract n t. – . . . • inclair , ao , as , as in , unha , a. lutamide nduced pospadias in ats ritical ssessment. l v , – . • a ebae , a pert e e ts , uc ouis , et al., ale eproductive isorders and ertilit rends nfluences of nvironment and enetic usceptibilit . l v. – . doi . ph srev. . .
#2020SOT 59 #toxexpo 59 Presenter 2 | PM14
• n n – • l – • l • • En l –
#2020SOT 60 #toxexpo 60 Presenter 3 | PM14
Postnatal Development of the Juvenile Male Reproductive Tract in Rats: Microscopic Evaluation, Interpretation, and Time Points of Toxicologic Significance
Catherine Picut, VMD, JD, DACVP, DABT, FIATP Charles River Durham, NC Phone: 919.619.0554 Email: [email protected]
1
Conflict of Interest Statement
The author declares no conflict of interest.
2
#2020SOT 61 #toxexpo Presenter 3 | PM14
Abbreviations • AMH: Anti Mullerian hormone • Rsp: round spermatid • DHT: dihydrotestosterone • SC: Sertoli cell SG: spermatogonia • Esp: elongating spermatid • • SP: spermatocyte • GATA: transcription sequence 5’-AGATAG-3 • T: testosterone • GD: gestation day • TDS: Testicular Dysgenesis Syndrome • HSD: hydroxysteroid dehydrogenase • TUNEL: terminal deoxynucleotidyl transferase • IHC: immunohistochemistry dUTP nick end labeling • VASA: protein encoded by vasa gene • LC: Leydig cell • MAGE-A4: melanoma associated antigen • MPW: masculinization programming window • OCT: Octamer binding protein • PCNA: proliferating cell nuclear antigen • PLAP: placenta-like alkaline phosphatase • PND: postnatal day
3
Introduction: Postnatal Stages of Development Stage Rat Human Neonatal Days 0–7 Days 0–1 Month Infantile Days 8–21 1 Month–2 Years Juvenile/Childhood Days 22–32 Years 2–11
Peripubertal Days 33–55 Years 11–14 Sexual Maturity Days 56–70 Years 14–16
Stages are approximate, as development occurs on a continuum
4
#2020SOT 62 #toxexpo Presenter 3 | PM14
Introduction We DOSE “Juvenile” Animals Often, but NO REGULATORY REQUIREMENT to Examine Immature Testes Microscopically
• Pubertal and Thyroid Function Assay in ntact u eni e eripu erta a e ats ui e ine 5 ose – 5 No requirement. • Juvenile Toxicity Studies ui ance or n ustr onc inica sa et e a uation o pe iatric ru ro ucts e ose u eni es ut e a uate en reac se ua aturit No requirement. • Nonclinical Safety Testing in Support of Development of Paediatric Medicines ar oni e ui e ines e a uate testes at se ua aturit No requirement. • Extended One-Generation (and Two-Generation) Reproductive Toxicity Studies an No requirement. • Rat Pre- and Postnatal Developmental Toxicity Study ui e ines 5 ection e pose t rou se ua aturation No requirement. • Reproduction/Developmental Toxicity Screening Test on ross e a ination o tissues at No requirement. • NTP’s Modified One-Generation (MOG) Reproduction Study— ose – No requirement.
5
Introduction “Juvenile” Testes Are More Commonly Evaluated in Investigatory Studies
• ost o at e a e earne a out to ic e ects on t e u eni e testis co es ro in esti ator stu ies one aca e ia an o ern ent researc epart ents e
• t a ates • isp eno • t er en ocrine isruptors or anoc orines
#2020SOT 63 #toxexpo Presenter 3 | PM14
ntrodu tion
Goal • To advo ate use of a uvenile sta e end oint in re lini al and environmental safety studies A enda • Microscopic features of the “juvenile” rat testis • Advantages of using “juvenile” testes as an endpoint Testi ular Dys enesis Syndrome Case Study E am les • Limitations of using “juvenile” testes as an endpoint • S e ial ro edures
Microscopic Features of “Juvenile” Testes
#2020SOT 64 #toxexpo Presenter 3 | PM14
at Testis eonatal
ell t pes
• mmature ertoli cells
• onoc tes
• Fetal Le dig cells L L niform tu ules
o lood Testis arrier igh Testosterone Levels
at Testis nfantile
ell T pes mmature ertoli cells permatogonia rogenitor Le dig cells L
Apoptosis
igh Mitotic ate L niform Tu ules
o lood Testis arrier
Lo Testosterone Levels
#2020SOT 65 #toxexpo Presenter 3 | PM14
at Testis Late nfantile
ell T pes • ertoli cells L • permatogonia
• permatoc tes
• mmature Le dig cells L
on uniform Tu ules Lumen forms L lood Testis arrier formed L Lo Testosterone Levels
at Testis Juvenile
sp ell T pes • ertoli cells • permatogonia • permatoc tes • permatids sp onl • Adult Le dig cells L
on niform Tu ules
L lood Testis arrier
ising Testosterone Levels
#2020SOT 66 #toxexpo #2020SOT
↑ Testosterone
testosterone ostnatal evelopment of Le dig ells T → → ncreaseTu ule Testes i e at T 67 67 Adapted from Lee et al • • J. ReprodFertil testosterone – – Presenter 3|PM14 #toxexpo Presenter 3 | PM14
First ave of permatogenesis in at Test’one
ertoli
Le dig
permatogonia
–
Advantages in Evaluating “Juvenile” Testes
#2020SOT 68 #toxexpo Presenter 3 | PM14
Advantages in Evaluating “Juvenile” Testes tential T i ants
i t – • • — • no “juvenile” endpoint •
Easil isi le ells
dult
#2020SOT 69 #toxexpo Presenter 3 | PM14
ni ue indings e sistent ent al n tes in at o ine in e n i ito iven
ont ol tini t t u io et l
ni ue T ansient indings e eased li e ati n e t li ells t l te iven t
l elin ont ol t l elin i et l
#2020SOT 70 #toxexpo Presenter 3 | PM14
ni ue indings ela ed atu ati n e t li ells Est adi l at
t n et l ont ol ≈PND 20 u t diol ≈PND 20
ni ue indings ela ed atu ati n i a e at tes et onid ole— –
ont ol i o e tene plenti ul eptotene otene nd lle di ete tu ule
#2020SOT 71 #toxexpo Presenter 3 | PM14
ni ue indings ultinu leated n tes in at
ni ue indings ultinu leated n tes in at
#2020SOT 72 #toxexpo Presenter 3 | PM14
ni ue indings Tu ule sgenesis in at
ni ue indings Tu ule sgenesis in at
#2020SOT 73 #toxexpo Presenter 3 | PM14
Testi ula sgenesis nd e n easingl n in an and li el due t envi n ental end ine dis u t s
A AT • • •
•
i s i eatu es Testi ula sgenesis nd e u an at
• • • in situ • •
• • • • • • • • • • •
#2020SOT 74 #toxexpo Presenter 3 | PM14
Tu ule sgenesis in at and an —
Tu ule sgenesis—Adult u an Testis
#2020SOT 75 #toxexpo Presenter 3 | PM14
In Situ
in situ
→
– –
#2020SOT 76 #toxexpo Presenter 3 | PM14
Juvenile Rat Testis “Transient”?
• — • — • •
Advantages in Evaluating “Juvenile” Testes
– • • — • •
#2020SOT 77 #toxexpo Presenter 3 | PM14
irst ave er at genesis in Rat igni i ant nu ers ra idl Test’one dividing and sus e ti le ell ulati ns ert li
e dig ‘Gonia
–
ase t ine a
• • – • • • — • — •
• •
#2020SOT 78 #toxexpo Presenter 3 | PM14
at Testes ont o “Mineral X”
—
i st a e e ion o nte est
#2020SOT 79 #toxexpo Presenter 3 | PM14
e Mea re ia e er Min r i
e ar e ia e er a le in e i
#2020SOT 80 #toxexpo Presenter 3 | PM14
a Ma e a ar ra r Mean a r
n l i n Mineral X e n e i
• • • The finding was considered a direct “Mineral X”– • The effect of “Mineral X” on the testes was transient and no longer present at PND 118, so including a “juvenile” endpoint allowed us to
—
#2020SOT 81 #toxexpo Presenter 3 | PM14
i i a i n in Evaluating “Juvenile” Testes
i itati ns in Evaluating Juvenile Testes
• ac ground findings that i ic to icit • “Degenerative” change in prepuberty • “Deficient” spermatogenesis in peripubertal phase • Don’t have all the cell populations available to you • od weight changes will effect a solute testis weights unli e in the adult • Translational issues • “Juvenile” rat is allegedly not a good odel for the infant childhood stage of an
#2020SOT 82 #toxexpo Presenter 3 | PM14
a g un egene ative in ings i i T i it poptosis of permatocytes at D ormal
Background “Degenerative” Findings Mimic Toxicity aucity of erminal pithelium an loughing at D ormal
#2020SOT 83 #toxexpo Presenter 3 | PM14
imitations Testes eig ts ange it Body Weight in “Juvenile” Male Rat
• n D rats • hen there is relate loss of bo y eight gain • here ecrease in absolute testes eights hen there is loss of bo y eight gain • here an increase in relative testis eights • herefore use absolute testes an epi i ymi es eights as in ication of to icity • o er absolute testis eights generally means testicular to icity
• n rats • hen there is relate loss of bo y eight gain • here a ecrease in absolute testis eights • here ecrease in relative testis eights • herefore use relative testes an epi i ymi es eights as in ication of to icity • o er relative testis eights generally means testicular to icity
an lati n et een Rat an u an tage Rat u an
ge vent ge vent e natal Days – Mini u e t Days – – hours n antile Days – Ra i onth– ears Mini u e t – li e ati n onths i u e uie en e onths– ears Juvenile Days – ears – hil h e i u e tal Days – ears – Ra i li e ati n l ule l ule
#2020SOT 84 #toxexpo Presenter 3 | PM14
e the e ie M e an lata le t Man
• • – •
• • – • –
e ial e u e
• ell e ti un hi t he i al tain γ β • • — •
#2020SOT 85 #toxexpo Presenter 3 | PM14
Rat e ti —
n al n te — u an e ti ea l tain
Front Endocrinol
#2020SOT 86 #toxexpo Presenter 3 | PM14
D’Andrea e a
D’Andrea e a
#2020SOT 87 #toxexpo Presenter 3 | PM14
• e na a n an e e n er n e e e n e e n and n rea ed en er a n an • n r ra n a a end n a D – a re ea ar e e a a er e nre n ed • Advantages outweigh limitations for including “juvenile” endpoint
• D – n ra ran a e ear – n an ere n ra rre a e e an e en er d n – ear • e a a n n n n n n a e ana are e e n e r evaluating “juvenile” testes
he nd
#2020SOT 88 #toxexpo Presenter 3 | PM14
• ampion et al ale eproto icit and ndocrine isruption EXS • or ier et al he eonatal estosterone urge A omparative tud ages eceived un u lished online ep https doi org • D’Andrea et al imultaneous A and a eling for esticular o icit valuation uggests hat etection of Apoptosis a e ore ensitive han roliferation Biotech Histochem • ostal A et al esticular o icit and educed ertoli ell um ers in eonatal ats i eth lhe l hthalate and the ecover of ertilit as Adults Toxicol and Appl Pharma • a tan et al ffects of eonatal strogen Administration on at estis evelopment with articular eference to ertoli ells J Androl • oei ansen et al arcinoma In Situ estis the rogenitor of esticular erm ell umours A linical eview Annals Oncology • oover et al ffects of efamandole on permatogenic evelopment of oung ats Tox Sci • uwada et al eonatal posure to ndocrine isruptors uppresses uvenile estis eight and teroidogenesis ut permatogenesis s onsidera l estored during u ert Bioch Biophy Res Comm
• i et al A ingle ose of i eth lhe l hthalate in eonatal ats Alters onoc tes educes ertoli ell roliferation and ecreases clin pression Toxicol and Appl Pharma • urmio et al nhi ition of rosine inases and it matini es late nterferes with ostnatal esticular evelopment in the at. Int J. Andrology https doi org j • ar et al esticular o icit of di eth lhe l phthalate in oung prague awle ats Toxicology • icut A et al eview omparative Aspects of re and ostnatal evelopment of the ale eproductive stem Birth Defects Research (B) • icut A et al ostnatal evelopment of the estis in the at orphologic tud and orrelation of orpholog to euroendocrine arameters Tox Pathol • a e ae et al esticular sgenesis ndrome An ncreasingl ommon evelopmental isorder with nvironmental Aspects Hum Reprod
#2020SOT 89 #toxexpo Presenter 4 | PM14
Pathology in Reproductive Toxicology Assessments and the Role of Stage-Awareness for Testis Evaluation
Cynthia Willson, MS, PhD, DVM, DACVP Integrated Laboratory Systems Inc. National Toxicology Program Pathologist (Contractor)/NIEHS Research Triangle Park, NC Phone: 984.287.3835 Email: [email protected]
1
Conflict of Interest Statement
The author declares no conflict of interest.
2
#2020SOT 90 #toxexpo Presenter 4 | PM14
Abbreviations
• ASG: accessory sex glands • H&E: hematoxylin and eosin • HRS: homogenization resistant spermatid count • MDF: Modified Davidson’s fixative • NBF: neutral buffered formalin • PAS/H: periodic acid Schiff/hematoxylin • RB: residual body • RT: room temperature • T: testosterone
3
Overview
• Holistic assessment of reproductive endpoints • Organ weights • Proper fixation and trimming of testis • Stage-awareness • Stages—basics • Why it’s important—something’s missing, something’s added, and stage-specific lesions • Patterns of toxicity in testis histopathology
4
#2020SOT 91 #toxexpo Presenter 4 | PM14
Holistic Assessment of Reproductive Endpoints
• Ferti ity f n tiona assessments—may e most sensitive for: • hanges in mating ehavior i ido, mo nting, ere tion, and e a ation • enotoxi effe ts transmissi e geneti a terations • resen e of agent dr g in semina f id • pigeneti hanges • perm parameters • ig de rease p to in some strains in sperm prod tion in rodents may have no dete ta e effe t on ferti ity • n ontrast, sma de rement in h man sperm o nt may a se inferti ity • Good histopatho ogy assessment is the most sensitive endpoint for the determination of testi ar toxi ity • ho d e done in on n tion ith epididymis, , and mammary g and • ndo rine organs, rain—a so affe t reprod tive f n tion • rgan eights—NEXT
Absolute or Relative Organ Weights?
Tissue Use absolute Notes Effect of ↓ body weight or relative? estis so te If absolute weight is ↓, should n ad t rats, no hanges in see histopatho ogy an have the eight or histopatho ogy signifi ant histopath findings of the testes or epididymides itho t a testis eight hange with ↓ in body weight gain of pididymis so te and If weight ↓ by 15% or more, ≤ 30% of controls. Mice and re ative should see ↓ sperm on yo ng rats, ho ever, are more histopatho ogy sensitive to ↓ body weight. rostate e ative rgan eight often more ↓ Body weight gains of ≥ 10% emina e ative sensitive than histopatho ogy of controls result in ↓ size esi e ery sensitive indi ator of and eight of the semina ins ffi ient androgens Weigh vesi es and prostate aref y ith se retions
hapin et a , and ehm et a ,
#2020SOT 92 #toxexpo Presenter 4 | PM14
Cheat Sheet to Changes in Organ Weights
Finding Look for Possible causes ↑ Testis weight ↑ Interstitial fluid (edema) ↓ Testis weight profiles d/t ↓ tubular fluid ↑ Epididymis weight ↑ Ductular fluid (edema) ↓ Resorption by rete, efferent ducts, initial segment ↑ Interstitial fluid (edema) ↓ Epididymis weight ↓ seminiferous fluid ↓ Prostate or seminal ↓ Serum T, steroidogenesis inhibition, androgen
INCREASED Testis Weight: Disturbance of Fluid Balance
Normal Seminiferous tubule dilation Interstitial edema ↑ Fluid in the seminiferous tubules ↑ Fluid in the interstitium alteration, ↓ lymphatic drainage DECREASED testis weight: disturbance of spermatogenesis (↓) +/- ↓ fluid production
#2020SOT 93 #toxexpo Presenter 4 | PM14
Appropriate Fixation, Trimming, and Staining
Tissue Fixation Trimming Staining Modified Davidson’s may miss the rete. Don’t 10% NBF for ≥48 hr Modified Davidson’s. 10% NBF for ≥48 hr 10% NBF for ≥48 hr at
Appropriate Trimming Efferent ducts of the Testis • – Rete Tubuli • testis recti rete testis tubuli recti Don’t confuse t ese t e e tu u es t t o c tu u es • n t t e ne e ss t e ete test s
#2020SOT 94 #toxexpo Presenter 4 | PM14
“Stage areness” in Microscopic Evaluation
• na es the dete tion of the most ommon mor ho o i a onse en e of in ry to ma e re rod tive system D M • e ard ess of tar et of to i ity • erto i e any one of the erm e o ations se ondary res onse to a tered hormone eve s a tered vas ar s y or a tered f id a an e • tho h most im ortant for shorter term st dies the testis re ard ess of st dy ty e sho d a ays e e amined ith an a areness of the s ermato eni y e • What is “stage awareness”? • ualitative not uantitative eva ation ased on fami iarity ith hat e asso iations are s osed to e in hat sta es • e ies on pattern recognition rather than o ntin sta es or e s • e ires some se of hi h ma nifi ation ≥
Is “Stage areness” Important?
• esti ar histo atho o y is onsidered the most sensitive arameter for the dete tion of testi ar to i ity • ensitivity in reases if the atho o ist assesses testi ar tiss e in a sta e a are manner • M st have s ffi ient no ed e of the a earan e of the erm e ty es ithin different sta e t es • Must no normal to appreciate a normal spermatogenesis • ther ise s t e t im ortant esions may e missed • e ative y easy to identify hen there are han es to the e s resent • More difficult to know when something is missing or shouldn’t be there! • “Stage awareness” allows for the ability to recognize: • hen an appropriate e ty e ayer is missing • hen an inappropriate e ty e is present • stage an or cell speci ic esions
#2020SOT 95 #toxexpo Presenter 4 | PM14
o o ou no I Stage areness as Implemente in a Screening Stu ?
• Pathologist should be requested to use “stage awareness,” not to “do testicular staging” • hould not be counting the number o ro iles in each stage • oo or ertinent language in the stud rotocol or re ort • Testes were evaluated in a “stage aware” manner . . . • detailed qualitative e amination o testes was made, ta ing into account stages o the s ermatogenic c cle . . . • icrosco ic evaluation was conducted to identi . . . missing germ cell la ers, retained s ermatids . . . • n cell or stage s eci icit o testicular lesions were noted . . . anning et al.,
Spermatogenesis
cle o S E M E ESIS com lete and ordered series o germ cell associations S ES in a given cross section o semini erous e ithelium over time. cle is wee s in rats.
#2020SOT 96 #toxexpo Presenter 4 | PM14
Spermatogenesis
Spermatogoniogenesis itoses and di erentiation o di loid s ermatogonia.
Spermatogenesis
Meiosis Tetra loid s ermatoc tes blue into ha loid round s ermatids in .
Spermatogoniogenesis itoses and di erentiation o di loid s ermatogonia.
#2020SOT 97 #toxexpo Presenter 4 | PM14
Spermatogenesis
Spermiogenesis Trans ormation o round s ermatids round cells w round nuclei into elongating elongated heads and long tails and inall mature s ermatids. ste s Meiosis Tetra loid in the rat shown here . s ermatoc tes blue into ha loid round s ermatids in .
Spermatogoniogenesis itoses and di erentiation o di loid s ermatogonia.
Spermatogenesis
Spermiation elease Spermiogenesis o mature ste Trans ormation o round elongated s erm rom s ermatids round cells the germinal e ithelium w round nuclei into into the lumen. elongating elongated heads and long tails and inall mature Meiosis Tetra loid s ermatids. ste s in s ermatoc tes blue the rat shown here . into ha loid round s ermatids in .
Spermatogoniogenesis itoses and di erentiation o di loid s ermatogonia.
#2020SOT 98 #toxexpo Presenter 4 | PM14
Stages o t e cle o Spermatogenesis
• S ES associations o germ cells rom di erent generations in concentric la ers that are alwa s seen together when loo ing at cross sections o tubules • These stages succeed each other over time and re eat to ma e u the c cle • o stages varies with s ecies. oman numeral stages I– I in the rat – in mouse . • S E a classi ication s stem o s ermatids based on the rogression o changes in s ermiogenesis • The ste s are based on the orm and sha e o the acrosome rom the olgi and, to a lesser e tent, the s ermatid head sha e and degree o chromatin condensation • ra ic numeral steps – in t e rat • stage is de ined b the oungest step o s ermatid, either roun or elongating
Stages an Steps? M Step T I– III have both I T I – I have onl roun and elongating elongating s ermatids, so s ermatids, so s ermatids o s ermatids o onl one ste two di erent ste s • ene fou ene t ons • ene fou ene t ons • s ermatogonia, • s ermatogonia, re ach tene s ermatoc tes, • ach tene s ermatoc tes, • • la ers o round s ermatids, • late ach tene s ermatoc tes, • elongating s ermatids • elongating s ermatids • Steps – P ste s – • te s – • The stage is named or the round I • tage te but oman s ermatid, which is a generation stage versus rabic ste ounger than the elongating s ermatid Step
#2020SOT 99 #toxexpo Presenter 4 | PM14
• – – n t e ouse • • – • • •
– –
#2020SOT 100 #toxexpo Presenter 4 | PM14
–
– – – –
– – – may “kiss” the Sertoli –
–
#2020SOT 101 #toxexpo Presenter 4 | PM14
yto lasmi elease Sta es ro lets small i e o elo ate s ermati s lei i e ter o yto lasm i ro Ste S s ermati s Sta e S t o – e eratio s yto lasmi ro ro lets lar e oarse o elo ati S elo ate s ermati s lei at asal ole Ste o yto lasm i ro s ermati s germ cell apoptosis – – Sta e
le s ith o e hromati lat o o e lo atio si e ro o other (“ sha e ”), RBs near lumen ro i Sta es a or es e i – Sta e le s ith o e hromati oth si es r e e tral a le ly elo ati and apex (“sesame s ermati s are Sta e see ”), RBs present near rese t ase or isa eari S ro i S S Sta e Sta es 2’ le s ith e tral a – orsal a le hromati condensing (“ a a a”)? lo atio Sta e spermatid retention Ski ier le s e se abnormal RBs chromatin (“ e t ro ho key sti k”)? Sta e
#2020SOT 102 #toxexpo Presenter 4 | PM14
heat heet to tages o the cle
• –VI (“Maturation”) • round • elongating • Stages VII and VIII (“Release”)— • line the lumen ith attached residual odies thinnest and “whorled” tails • • •
2
heat heet to tages o the cle
• –XII (“Elongation”)— • • shape o the elongating spermatid head • Stage XI: nucleus asymmetric “D shaped” (flat on one side); open, pale chromatin + RBs present in • Stage X: nucleus “sesame seed” shaped with ventral “V”; open, pale chromatin; RBs gone or basal • Stage XI: nucleus “banana” shaped, chromatin condensing at base of nucleus • Stages XII and XIII: thinning “bent rod” or “hockey stick” nucleus, dense chromatin • Stages XIII and XIV (“Grouping”) • huge diplotene spermatoc tes an meiotic igures secondar spermatoc tes •
2
#2020SOT 103 #toxexpo Presenter 4 | PM14
Look for the “Helpers”
• he testis is not like Vegas • hat happens in the testis usually does stay in the testis • hanges in sperm parameters often will be linked to changes in histopathology • ↑ in HRS? Look for spermatid retention in the testis. • ↓ in HRS? Look for ↓ elongated spermatids in the testis. • esticular to icity is often reflected in cellular, fluid, or other changes in downstream, androgen dependent, or endocrine tissues • E g , debris e foliated germ cells in the epididymis igure out where they came from • ook for weight and or histopathological changes in: • Epididymis, accessory se glands, pituitary gland, mammary gland (male rats)
hree e so s h t e re ess s port t
• ( ) hen an ppropr te cell type layer is ss • It is relatively easy to identify when there are changes to the cells present • More difficult to detect when something is missing • E amples: depletion of spermatocytes, depletion of elongating spermatids • ( ) hen an ppropr te cell type is prese t • E ample: spermatid retention in stages IX–XII (also, a stage specific lesion) • ( ) o identify st e or ell spe f lesions • E ample: germ cell apoptosis in stages VII VIII
#2020SOT 104 #toxexpo Presenter 4 | PM14
s t or l
he ppropr te ell pe L er s ss eplet o of per to tes
• • •
#2020SOT 105 #toxexpo Presenter 4 | PM14
he ppropr te ell pe L er s ss eplet o of lo t per t s
contracted in diameter (due to loss of elongating spermatids and likely due to ↓ T). Germ cell depletion is the “Helpers”: ↓HRS; may see ↓ testis weight (↓ spermatids and fluid) and ↓ epididymis weight/sperm/fluid.
he ppropr te ell pe s rese t per t ete t o —A “Hallmark” Stage pe f Les o • • – • • • • • • • “Helpers” • • • • If ↑ HRS, be sure to look for spermatid retention!
#2020SOT 106 #toxexpo Presenter 4 | PM14
S ermat ete t A Stage S e e –
Ste
Ste Ste Ste
r l s e le s ep sper s r e le re e s ep sper s e s ep r l l per rre s e e l e e ppr pr e s ep sper s sper s er re e s er e es s eep e r ep el
am le arl ell a Stage S e t
em al r mar ell erm ell a e te Stage e t e ere e target e e l e e e e sper e – l se re s s er sper e e – – se re s e l ll s es se r e e e er l ell e es e – – e l r e l per re e – re e Hess e l He e e er l ell e es e – e el e e sper s e p e l Elongating s’tids (death) e e e e ell e es e r le e l s lp e ep sper s e err e l • e spe r se r • ere ell r e s res l p e e sper e e p p s s • e s e e ls res l p e e r sper p p s s s l e s e e r r re s
#2020SOT 107 #toxexpo Presenter 4 | PM14
• eat es • to las i eosino hilia and ont a tion • h o atin ondensation (es s e ato tes) o a gination (es o nd s e atids) • Note: don’t confuse with residual bodies in stage VIII– or low le el bac ground attrition II IV • e ell a o tosis an o di e tl (da age to ge ells) o indi e tl (da age to so ati ells e toli o e dig ) ( oe elheide ) • • (int atesti la no all se ) • hen esent seen in E lo n e s o t les ells so e i es a e l e a ination • single in e tion o ethane di ethane s l onate a te th ee da s e dig ell o lite ation and ndete ta le testi la t still onl lo n e s o stage t les ith a o toti s e ato tes ( a tlett et al ) • onside ed the ost sensiti e a e o ed ed and an ea l e e t o and ogen de i ien ( ssell et al )
eas eas et al
tage t le o a at de onst ating n e o s a o toti a h tene s e ato tes (a o s) onsistent ith ed ed testoste one ( E)
Note: this lesion has also been obser ed in stage VII ach tene s er atoc tes in oung rats in short ter food restriction studies ( eh et al )
#2020SOT 108 #toxexpo Presenter 4 | PM14
• o e hanges a e non stage s e i i • e toli ell to i it o ten eat es se e al nons e i i degene ati e lesions • esions a sta t o t s e i i and end in nons e i i degene ation and depletion → atrophy • o e a les eat ing stage and o ell s e i i hanges • nd ogen de i ation • Ea l hanges • o stage s e i i lesions a h tene a o tosis stage s e atid etention • og essi e hanges—de letion o elongating s e atids • “Helpers”— it ita gland ale at a a gland • Est ogeni o o nds in ale ats • i ila lesions as de eased testoste one in the testis • “Helpers”— ale at a a gland
• o e hanges a e non stage s e i i • e toli ell to i it o ten eat es se e al nons e i i degene ati e lesions • esions a sta t o t s e i i and end in nons e i i degene ation and depletion → atrophy • o e a les eat ing stage and o ell s e i i hanges • nd ogen de i ation • Ea l hanges • o stage s e i i lesions a h tene a o tosis stage s e atid etention • og essi e hanges—de letion o elongating s e atids • “Helpers” – it ita gland ale at a a gland • Est ogeni o o nds in ale ats • i ila lesions as de eased testoste one in the testis • “Helpers”— ale at a a gland
#2020SOT 109 #toxexpo Presenter 4 | PM14
• —“easy to injure, hard to kill” • • • “micro ” or “ ” • • Focal germ cell “dropout” • • • • The exfoliated cells look rounded and “normal” and lack degenerative features • — • • • • • • —
—
#2020SOT 110 #toxexpo Presenter 4 | PM14
rogre e ect o ertol ell ur
acuolation
erm cell dro out eneral degeneration ith germinal e ithelium disorgani ation as ell as de letion and degeneration of elongating egeneration s ermatids These of elongating effects are not stage s ermatids or germ cell s ecific
atter o o c t • ome changes are non stage s ecific • ertoli cell toxicity often features several nons ecific degenerative lesions • esions may start out s ecific and end in nons ecific degeneration and depletion → atrophy • T o exam les featuring stage and or cell s ecific changes • ndrogen de rivation • arly changes • T o stage s ecific lesions achytene a o tosis stage , s ermatid retention • rogressive changes—de letion of elongating s ermatids • “Helpers”— , ituitary gland, male rat mammary gland • strogenic com ounds in male rats • imilar lesions as decreased testosterone in the testis • “Helpers”— , male rat mammary gland
#2020SOT 111 #toxexpo Presenter 4 | PM14
o trol atter o o c t o • • • apopto pac te e permatoc te tage • permat d rete t o • • reated t a p t alate or t o ear • ecrea ed te t e g t • rogre e dege erat o a d deplet o o elo gat g permat d • fluid, so depletion of elongating spermatids → decreased fluid → decrea ed tu ular d ameter • educed ep d d m e g t a d po perm a • ecrea ed pro tate em al e cle e g t tolog cal atrop “Gonadectomy cells” in • Pituitary gland pars distalis “castration cells” t e p tu tar gla d par d tal
atter o o c t troge
• • Also, “mimic” T in the negative feedback on GnRH and LH release → ↓ LH → ↓ • ect o e troge te t ca loo m lar to lo • • – • •
eft ight to left center
#2020SOT 112 #toxexpo Presenter 4 | PM14
ont ol atte ns o o icity o e s s st o ens Look for the “Helpers” • Estrogenic agent “helpers”: • o solate al ostate es onse to est o en ac te ne t o ilic in lammation • ale rat a ar glan — ale to fe ale ifferentiation
eated it a • Low T “helpers”: ste oido enesis in i ito • ent al ostate es onse to lo and o en at o y lo e ost sensiti e to circ lating an rogen le els • ecrease weights icroscopic atroph : epi i is prostate se inal esicle • ↓ weight may be more sensitive than histopathology • ale rat a ar glan atroph
t o y o ent al lo es o Creasy, 2008; O’Connor et al., 2002; Tang anl ekal an o inette t e ostate land
mma y and oncl sion
• Testes sho l e e al ate in a stage aware anner • To detect what’s missing, what’s there that shouldn’t be there, or stage specific lesions
• Take the holistic iew • Look for the “helpers” • ntegrate the en points a aila le weights histopatholog of repro cti e an en ocrine tiss es sper para eters fertilit • E a ine repro cti e an en ocrine organs as a nit to e al ate patterns of change
#2020SOT 113 #toxexpo Presenter 4 | PM14
efere es • artlett err an harpe The Effect of electi e estr ction an egeneration of at Le ig ells on the ntratestic lar istri tion of Testosterone an orpholog of the e inifero s Epitheli ndrol : • lack rn ra Llo hear an oster o parison of the Effects of the Three so ers of initro en ene on the Testis in the at o icol l har acol : • oekelhei e at Testis ring he ane ione nto ication an eco er ose esponse an the e ersi ilit of er ell Loss o icol l har acol : • oekelhei e echanis s of To ic a age to per atogenesis Natl ancer Inst onogr : – • hapin E organ T an s The orphogenesis of Testic lar egeneration n ce in ats rall inistere he ane ione olec athol : • hapin E lati arnes L Teag e L The Effects of ee estriction on epro cti e nction in prag e awle rats unda l o icol : • ler ont an ere The tages of the cle of the e inifero s Epitheli of the at: ractical efinitions in chiff e ato lin an e ato lin eosin taine ections e anad iol : • ook ohnson L onnor iegel L ra s ra e an rtt E Effects of ietar eta Estra iol E pos re on er or one oncentrations an Testic lar ara eters in ale rl: ats o icol ci :
efere es
• reas E al ation of Testic lar To icit in afet E al ation t ies: The ppropriate se of per atogenic taging o icol athol : • reas athogenesis of ale epro cti e ste To icit o icol athol : • reas En ocrine isr ption: i ance oc ent for istologic E al ation of En ocrine an epro cti e Tests art : ale repro cti e s ste http: www oec org ataoec p f art : a ar glan http: www oec org ataoec p f art : it itar glan http: www oec org ataoec p f rgani ation of Econo ic ooperation an e elop ent • reas e e i k E an ori wahara asson olte T ea s egan eh ogerson hitne roliferati e an onproliferati e Lesions of the at an o se ale epro cti e ste o icol athol : • reas l nn ra T an tler antitati e t of tage pecific per atoc te a age following inistration of Eth lene l col ono eth l Ether in the at olec athol : • reas an oster The orphological e elop ent of gl col ether in ce testic lar atroph in the rat olec athol : • ess Lin er E tra er L an errea lt c te Effects an Long Ter e alae of initro en ene on ale epro ction in the at antitati e an alitati e istopatholog of the Testis ndrol : • err illar a ocks an harpe tage epen ent hanges in per atogenesis an ertoli ells in elation to the nset of per atogenic ail re following ith rawal of Testosterone nat ec :
#2020SOT 114 #toxexpo Presenter 4 | PM14
efere es
• o icol athol – • nn N cad ci • O’Connor, JC, Frame SR, and o icol ci • o icol athol • – • er ells and ertili ation • ndocrinolog • er atogenesis • rch o icol
#2020SOT 115 #toxexpo Update your skills with CEd-Tox: SOT Continuing Education Online
Why CEd-Tox? Stay competitive and keep Learn anywhere, your knowledge up-to-date anytime in your field Access presentations by Earn hours for top experts certification
Diverse course offerings View in a group cover a range of topics setting
Enroll at www.toxicology.org/cedtox
Student and Postdoctoral SOT members, SOT Undergraduate Affi liates, and individuals from developing countries receive FREE access. Bring new research and perspectives to the 2021 SOT Annual Meeting. Submit a Scientific Session or Continuing Education (CE) course proposal.
Proposals are due May 15, 2020. www.toxicology.org/2021