Transcript 정소독성 평가기법

정소독성 평가
Evaluation of testis toxicity
산업안전보건연구원
화학물질안전보건센터
정용현
042-869-0345
[email protected]
Testis
Function
– Production of male gametes
– Production of male sex hormones
(testosterone)
Location
– Within the scrotum of most mammals
• 3~5 ℃ cooler than body temperature
– Parenchyma (capsule)
Testis (structure)
• Closely packed seminiferous tubules of
200~250 microns in diameter
• Human : separated by fascia (근막)
• Rodents : no subdivisions
– Rat : 30 tubules / testis
1mm (length) / each tubule
Testis (structure)
• Interstitium (정세관과 정세관 사이)
– Blood and lymph vessels
– Macrophages
– Leydig cells (testosterone)
• Rete testis (정소망)
– Ends of the seminiferous tubules
– Intercommunicating system of channels lined with
a low cuboidal/columnar epithelium
– Upper pole of the testis (rat)
Gametogenesis(배우자형성)
• The process of formation and development of
gametes (germ cells)
– Mitosis, meiosis and cell shape changes
• Seminiferous tubules
– Sertoli cells
– Developing germ cells
• Spermatogonia, primary spermatocytes, secondary
spermatocytes, spermatid, spermatozoa
– Myoid cells
• Aid the movement of spermatozoa and fluid along the
lumen
Gametogenesis
• Spermatogenesis (정조세포 -> 정모세포)
• Spermiogenesis (정자세포 -> 정자)
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Spermatids are reshaped into spermatozoa
Acrosome
Nuclear material condenses
A mid piece forms with aggregation of
mitochondria
– A flagellum is generated (편모형성)
– Shedding of excess cytoplasm (허물)
Sertoli cells
spermatogenesis
spermiogenesis
• `Nurse’ cells
• Regulation of
and
– Structural support
– Provision of a specialized luminal fluid
environment
– Nutrition
– Phagocytosis
– Development and release of spermatozoa
Blood-testis barrier
• Sertoli-Sertoli cell barrier
– Specialized luminal fluid environment
– Protect spermatozoa from the immune system
and harmful substances
– Sertoli-Sertoli tight junctions
– The barrier divides the seminiferous epithelium
into 2 (basal and adluminal) compartment
The Epididymis
• Abandoned child of the reproductive system
– Current : Motility and Morphology
– Future : Sperm Proteins, Fertilization Assays
• Sperm Maturation in the Epididymis
: Functional Change
- Sperm acquire motility
- Sperm acquire the ability to fertilize an oocyte
Requires interaction between sperm, epididymal
epithelium and luminal fluids
Origin of Adverse Effects on
Epididymal Sperm Function
• Testicular
• Direct on epididymal spermatozoa
• Direct on epididymal epithelium
• Epididymal Toxicants
• Methyl Chloride,
• Alpha-chlorohydrin
• Alkane sulfonates
(Few chemicals)
Epididymis (Structure)
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•
•
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•
•
•
Efferent ducts
Initial segment
Caput (head)
Corpus (body)
Cauda (tail)
Distal
Deferens
Fertilization
• Spermatogenesis
– Testis
• Maturation
– Epididymis
• Fertilization
– Female Reproductive Tract
Movement
• Fluid secreted by the Sertoli cells and the released
spermatozoa pass along the lumen of the
seminiferous tubule, into the rete testis, the
efferent ducts and into the epididymis.
• Rate of movement within the lumen of a rat
seminiferous tubule : 1㎕/hour
• Myoid cells and Sertoli cells
• At this stage, spermatozoa are not able to swim.
Efferent ducts
• 6 efferent ducts
a single duct
epididymis (rat, mouse)
join to form
before entering into the
Efferent ducts
• Function
– Reabsorption of water
(90% of fluid leaving the testis)
– Spermatozoa
within the efferent ducts of the rat are
concentrated (3%~20%) of the total volume
Epididymal duct
• Pseudostratified columnar epithelium
• Cells contain a brush border and large Golgi
apparatus
• Surrounding the tubule is a muscular wall that
thickens from proximal to distal regions.
Epididymal cell types
• Principal
– Proximal resion > distal resion
– Secretion and absorption of macromolecules, ions, organic
solutes
• Narrow
• Clear
– Absorption of macromolecules
• Basal
– Protection of epithelium, spermatozoa from xenobiotics
• Halo
– May be Monocytes or lymphocytes
Changes in the epithelium
• From
proximal to distal
regions
– The height of epithelium is reduced
– The luminal diameter increases
– Appearance of different cell types
– Presence or absence of stereocillia
– Increase in the degree of smooth muscle
surrounding the duct
Blood-Epididymis Barrier
• Among the various epithelial cell contents examined,
the occlusion of the
developed.
• Tight junction
spaces.
epididymis
is the most highly
separate the basal and luminal
Luminal Fluid Microenvironment
• The composition changes dramatically from
proximal to distal regions.
• The composition is the result of secretion and
absorption of molecules across the epididymal
epithelium.
• Changing environment is responsible for changes
that occur within spermatozoa as they progress
along the epididymal duct
Sperm Maturation
• Spermatozoon (pl. spermatozoa)
• The relative position along the epididymal duct for
varies species where spermatozoa acquire the
ability to fertilize an egg.
– rabbit (corpus)
– mouse, rat, man (cauda)
Changes in Spermatozoa During
Epididymal Transit
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Surface charge
Lectin-binding properties
Nuclear bonds
Lipid and carbohydrate composition
Membrane fluidity
Surface proteins
• Many of these changes continue through
ejaculation, acrosome reaction, fertilization
Sperm transport (days)
5
Monkey
Rabbit
Rat
Man
10
15
Sperm Storage
• Sp. In Cauda (x 1,000,000) / Daily Sp. Production
Man
3
Rat
5
Rabbit
10
Sperm Protection
• Spermatozoa must be protected from the
immune system, harmful xenobiotics and
agents that may cause oxidative stress.
• The blood-epididymis barrier
role in the protection of sperm.
plays a major
정소독성평가
1)
2)
3)
4)
5)
6)
정소 육안관찰 (장기무게, atrophy)
정세관 전체 관찰 (tubular necrosis)
정세관내 생식세포 관찰 (spermatogenesis)
Sertoli cells 관찰
Leydig cells 관찰
정소상체 : caput, corpus, cauda 관찰
–
–
Aberrant cell types in lumen
Absence of clear cells in cauda
Germ cells
• Assesment of testicular degeneration
(a) Subjective assessment using modifiers
- mild, moderate, severe
(b) Measurement of the frequency of abnormal
tubules
(c) No. of XIV stage / No. of the total tubules
(d) Selected degrees of tubular abnormality
severe degrees = Classify the eight grades of
degeneration x No. of tubules
(e) Sertoli cell index (SCI)
SCI = No. of germinal cells / No. of Sertoli cells
정조, 정모세포
• stage II-III
• 정조세포의 괴사
• Sertoli cell의 탐식작용
• stage VII
– (정조세포, preleptotene, pachytene, 정자세포)
• 정조세포, preleptotene 정모세포 소실
• 정세관 기저층에 pachytene 정모세포
정자세포
• Stage IX
• Sertoli cell의 장해 (* 공포형성)
• 정세관의 기저부에 정자세포 (step 19)가 박리되
지 않고 잔류 되어 있다.
• Stage VI
• 정자세포의 다핵거대화
• Sertoli cell 장해에 따른 변화
Tubular necrosis
• Tubular necrosis occurs as a results of ischemia
• Cadmium (testicular blood supply)
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cadmium 단회 투여 후 2일차 (rat)
혈관내피 장해
ischemia (허혈), 순환장해
정소의 경색상 변화
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cadmium 단회 투여 후 7일차 (rat)
정세관 괴사
간질의 염증 반응
stage 특이성, 세포 특이성이 보이지 않는다.
Sertoli cells
• Sertoli cells do not divided after 18-21 days of age.
• Structural support and movement of germ cells.
* 19 spermatid retention at VIII stage
• Phagocytic activity.
• Formation of the blood-tubule barrier
• Cellular metabolism and intercellular interactions
• Sertoli-only syndrome
– Ethanol 단회 투여 후 14일차 (rat)
– 생식세포의 소실
Leydig cells
-
testosterone.
Large pale cells in the pituitary known as castration
cells are often found in association with
severe bilateral testicular atrophy and Leydig
cell hyperplasia.
The major site for the synthesis of
-These are presumed to secrete gonadotropic
hormones.
Leydig cell loss
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•
•
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Ethanol 단회 투여 후 7일차 (rat)
Leydig cell 소실
Testosterone 생산저하
정자세포, 정모세포 의 변성 및 괴사
Leydig cell hyperplasia
• Leydig cells 증가
• Pituitary 내의 castration cells 출현
– Leydig cell 의 testosterone 분비 기능조절은
pituitary에서 분비되는 LH 에 의하여 조절된다.
– Testosterone levels 은 pituitary 의 LH 분비를 조절한다.
• Leydig cell 의 testosterone 분비기능 이상은
pituitary의 LH 분비를 자극하여
Leydig cell hyperplasia를 초래한다.
Finding of primary target cell
-Time course study of spermatogenesis
-Maturation depletion :
The progressive loss of subsequent cell
types following injury to a precursor
cell
표적세포
• 투여기간, 투여농도에 따라
표적세포는 달라질 수
있다.
• 생식세포는
세르토리세포에 의하여 24시간 내에 탐
식된다.
• 정자세포는 정세관 내강으로 계속 빠져나가므로 표
적세포를 찾는데 혼란스러울 수 있다.
스테이지의 각 생식세포의 종류별 수 비교
• 통계적 유의성 (SCI) : 표적세포
• 동일한
정자형성과정
- 14 different stages
of cellular association
: 19 different steps of spermatid (acrosome structure)
- Rat : 48-53 days
(a cycle time of 12-13.3 days)
- 스테이지 별로 소요되는 시간 :
• 각 스테이지마다 다르며
• 정소에서 출현하는 정세관 스테이지 빈도수와 비례하게 된다
(Haschek and Rousseaux 1991).
Table 1. Body weight (g) and relative testes weight (mg) of male rats treated with 2bromopropane for 28 days
Control
125 mg/kg
250 mg/kg
500 mg/kg
Initial body
weight
300.5 ± 14.3
300.7 ± 12.7
300.3 ± 12.7
301.4 ± 13.2
Terminal body
weight
356.7 ± 16.1
352.5 ± 11.8
330.4 ± 10.7*
289.8 ± 16.0*
Testes (L)
475.5 ± 30.7
467.9 ± 36.7
402.9 ± 49.3*
329.8 ± 88.8*
Values are means ± S.D.
Significantly different from control group at * p<0.05 or ** p<0.01.
Relative testes weight = (testes weight / terminal body weight) x 100g.
L: Left.
Table 2. Numbers of Sertoli cells and Sertoli cells indices (SCI) of rats treated with
2-bromopropane for 28 days
No. of
cells
Sertoli
SCI
Control
125 mg/kg
250 mg/kg
500 mg/kg
17.7 ± 2.2
16.8 ± 1.2
13.6 ± 1.0*
16.1 ± 1.8
30.2 ± 2.8
26.9 ± 4.7
21.0 ± 3.0**
11.8 ± 1.4**
Values are means ± S.D.
Significantly different from control group at * p<0.05 or ** p<0.01.
SCI = Total number of germ cells / total number of Sertoli cells.
Total numbers of testes in each group = 6.
Number of tubules in each testis = 13.
Table 3. Sertoli cells indices (SCI) of each spermatogenic cell of rats treated with 2bromopropane for 28 days
Control
125 mg/kg
250 mg/kg
500 mg/kg
1.2 ± 0.1
1.0 ± 0.1*
0.4 ± 0.2**
0.3 ± 0.1**
preleptotene
3.6 ± 0.6
2.6 ± 0.5**
1.0 ± 0.2**
0.0 ± 0.0**
leptotene
4.1 ± 0.7
3.2 ± 0.5
1.6 ± 0.2**
0.2 ± 0.1**
zygotene
4.6 ± 0.7
3.9 ± 0.6
1.5 ± 0.6**
0.0 ± 0.0**
pachytene
4.7 ± 0.6
3.8 ± 0.3**
2.0 ± 0.9**
0.0 ± 0.0**
diplotene
5.9 ± 1.1
5.9 ± 1.2
2.5 ± 2.1*
0.0 ± 0.0**
round
15.1 ± 1.4
12.7 ± 0.7*
9.8 ± 2.2**
1.8 ± 0.6**
elongate
12.1 ± 1.1
11.0 ± 1.0
12.1 ± 0.9
10.4 ± 1.4*
SCI
Spermatogonia
Spermatocyte
Spermatid
Values are means ± S.D.
Significantly different from control group at * p<0.05 or ** p<0.01.
SCI = each number of germ cells / total number of Sertoli cells.
Total numbers of testes in each group = 6.
Number of tubules in each testis = 13.
Table 4. At indicated stage, Sertoi cell indices (SCI) % of each germ cell of rats treated
with 2-Bromopropane 125 mg/kg for 28 days
stage
spermatocytes
spermatogonia
preleptotene
leptotene
zygotene
spermatids
pachytene
diplotene
round
elongate
I
100±25
79±20
87±34
94±43
II-III
54±11**
92±44
94±44
100±37
IV
108±31
99±26
99±23
102±17
V
83±40
78±16*
84±28
90±30
VI
78±9**
85±11*
89±10*
94±25
VII
104±53
68±28
83±44
72±25
56±22
VIII
90±30
56±35*
75±29
77±29
82±33
IX
70±45
89±28
88±36
104±41
X
50±18**
73±27*
80±27
98±13
XI
66±27*
86±18
101±30
89±27
XII
112±37
82±14*
92±21
95±22
XIII
66±19*
87±28
XIV
95±59
83±35
102±30
104±20
103±38
98±29
Values are means ± S.D. Significantly different from control group at * p<0.05 or ** p<0.01.
SCI = The number of the each germinal cells / total number of Sertoli cells.
Date are expressed as % of control value. Total numbers of testes = 6. Number of tubules in each testis = 13.
표적세포
• spermatogonia, spermatocytes,초기 spermatids 에서
대조군에 비하여 통계적으로 유의한 생식세포의 감소.
•
시험물질이 spermatogonia에 영향을 미쳐 4주 후,
spermatogonia, spermatocytes, 초기 spermatids의 손
실로 나타난 것이다.
• 만일 spermatocytes에 영향을 미쳤다면,
spermatocytes와 후기 spermatids의 감소가 나타났을
것이고, spermatogonia의 수는 변화가 없었을 것이다.
•
결론적으로 정자발생과정과 세르토리세포의 탐식작용
을 고려할 때, 시험물질의 표적세포는 spermatogonia.
정소독성변화의 해석
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•
•
•
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생식세포중 표적세포의 추정 (Ettlin, 1984)
단회 투여 (rat)
종축은 투여 후 주, 횡축은 각종 생식세포
종축의 각종 생식세포의 간격은 정자형성 cycle
(spermatogenesis) 진행시간을 고려한 것임.
예 : 단회투여 후, 3주 후에 부검하여 zygotene 정모세포 감소
* 투여 후 3주 의 횡축의 zyrotene 정모세포의 위치에 점을 찍고,
* 챠트의 사선과 평행한 선을 아래쪽으로 그어 보면,
* 그 선의 선단은 정조세포 type A와 만난다.
* 이 세포가 표적세포로 추정된다.
표본제작
1. 고정 : Bouin 용액에 고정 (약 24시간 이내)
1) 2-3시간 고정
2) 침투를 양호하게 한 후
가운데 부근에 횡단할면을 넣어 재고정
3) 약 12시간 후 3-4mm의 두께로 절취, 재고정
2. 절취 :
1) 좌우의 정소를 횡단하고 3-4mm 두께
2) 정소상체 : 종(longitudinal section) 절취
3. 조직처리
4. 포매
5. 박절
6. 염색 : PAS
1) 탈파라핀, 함수
2) 0.5% Periodic acid 20분
3) D.W 수세
4) Schiff reagent 30분
5) 흐르는 물에 수세
6) Hematoxylin 대조염색 15분
7) 흐르는 물에 수세
8) 탈수
9) 투명
10) 봉입