TESTICULAR BLOOD FLOW
Methological and functional studies in the ra t av
Jan-Erik Damber la'kare
In stitutionen fö r Fysiologi, Umeå U n iversitet, S-901 87 Umeå
Akademisk avhandling som med vederbörligt tills tå n d av Medicinska Fakulteten vid Umeå U niversitet för avläggande av medicine
doktorsexamen kommer a tt framläggas fö r o ffe n tlig granskning i sal C, universitetsbyggnad LU-0, Umeå U n iversitet, fredagen den 19 maj kl 10.00.
Avhandlingen baseras på följande rapporter:
I Methodological aspects of te s tic u la r blood flow measurements in rats. J .-E . Damber and P.O. Janson: Acta physiol, scand.
1977. 101. 278 - 285.
I I Testicular blood flow and testosterone concentrations in spermatic venous blood of anaesthetized ra ts . J .-E . Damber and P.O. Janson: J. Reprod. Fert. 1978. 52. 265 - 269.
I I I The effects of LH, epinephrine and norepinephrine on t e s t i
cular blood flow and plasma testosterone concentrations in anaesthetized ra ts . J .-E . Damber and P.O. Janson: Acta endo- c rin o l. (Copenh.), accepted fo r publication 1978.
IV Testicular blood flow and testosterone concentrations in the spermatic venous blood in rats with experimental cryptor
chidism. J .-E . Damber, A. Bergh and P.O. Janson: Acta endo- c rin o l. (Copenh.), accepted for publication 1978.
V The influence of scrotal warming on te s tic u la r blood flow and endocrine function in the ra t. J .-E . Damber and P.O.
Janson: Acta physiol, scand., accepted for publication 1978.
DAMBER, J .- E ., T esticular Blood Flow. Methodological and functional studies in the r a t. Umeå Univ Med Diss, New Series, 39, 1 - 43 , 1978.
D iffe re n t methods of measuring te s tic u la r blood flow in the ra t were compared in an attempt to find an accurate method fo r measuring physiological te s tic u la r blood flow. I t was found that both the Xenon- 133 clearance technique and the radioactive microsphere technique probably r e fle c t true physiological blood flow in the te s tis .
The microsphere method was used to study some functional aspects of te s tic u la r blood flow. There was a s ig n ific a n t positive co rrela
tion between the te s tic u la r blood flow and the outflow of testosterone in the spermatic vein, indicating that te s tic u la r hormone secretion may be affected in d ire c tly via a primary e ffe c t on te s tic u la r blood flow. In tr a -a r te r ia l infusion of LH caused a s ig n ific a n t decrease in the vascular resistance of the te s tis . However, the e ffe c t was small in comparison with the simultaneous e ffe c t of LH on plasma testosterone concentration, indicating that blood flow changes are not c r i t ic a lly involved in the acute e ffe c t of LH on te s tic u la r endocrine function.
Infusion of epinephrine or norepinephrine did not induce any absolute changes in te s tic u la r blood flow, but norepinephrine caused an in crease in te s tic u la r vascular resistance. Both catecholamines caused s ig n ific a n t depressions in plasma testosterone concentration. I t was concluded that the catecholamine induced reductions in testosterone concentration were not due to a vascular e ffe c t on the te s tis .
Testicular blood flow and Leydig ce ll function in the cryptorchid and heated te s tis were also studied. There was a s ig n ific a n t increase in re la tiv e blood flow in the cryptorchid te s tis , probably due to an highly altered morphology consisting of a re la tiv e increase in in te r s t i t i a l tissue containing blood vessels. Furthermore, i t was found that the testosterone le v e ls , in spermatic vein blood from the cryptor
chid te s tis , were highly reduced in comparison to the corresponding values fo r the scrotal te s tis and the outflow of testosterone from the cryptorchid te s tis was estimated to be only 13% of that from the scrotal one. This re s u lt suggested that the Leydig c e ll function was greatly impaired in the cryptorchid te s tis . The vasculature of the te s tis is re la tiv e ly insensitive to local heating since no effects on vascular resistance were observed when warming the te s tis to ab
dominal temperature. On the other hand, there was a s ig n ific a n t in crease in blood flow in the te s tis a t 41 and 43° C, which are tempera
tures known to induce cessation of spermatogenesis in the r a t. The acute response of the te s tis to LH stimulation was reduced when warm
ing the scrotum to 41 and 43° C. This strongly indicates an impaired Leydig cell function a t these temperatures. Since blood flow was in creased at these temperatures i t was concluded that the reduced Leydig c e ll responsiveness to LH was unrelated to te s tic u la r perfusion.
Jan-Erik Damber
Dept of Physiology, Univ. Umeå, S-901 87 Umeå, Sweden
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 39
From the Department of Physiology. University of Umeå, Umeå, Sweden
TESTICULAR BLOOD FLOW
Methodological and
Functional Studies in the Rat
Jan-Erik Damber
Umeå Universitet Umeå 1978
LIST OF PUBLICATIONS
The present th e s is is based on the f o llo w in g papers, which w i l l be r e f e r r e d to in the t e x t by t h e i r Roman numerals.
I Methodological aspects o f t e s t i c u l a r blood flow measurements in r a t s . J . - E . Damber and P.O. Janson: Acta p h y s io l, scand.
1977. 101. 278 - 285.
I I T e s t i c u l a r blood flow and tes to s te ro n e conce ntra tions in spermatic venous blood o f an aesth etized r a t s . J . - E . Damber and P.O. Janson: J. Reprod. F e r t . 1978. 52. 265 - 269.
I I I The e f f e c t s o f LH, epinephrin e and norepinephrine on t e s t i c u l a r blood flo w and plasma tes to s te ro n e conce ntra tions in an ae sth etized r a t s . J . - E . Damber and P.O. Janson: Acta endo- c r i n o l . (Copenh.), accepted f o r p u b l ic a t i o n 1978.
IV T e s t i c u l a r blood flow and te s to s te ro n e concentra tions in the spermatic venous blood in r a t s w ith experimental c r y p t o r chidism. J . - E . Damber, A. Bergh and P.O. Janson: Acta endo- c r i n o l . (Copenh.), accepted f o r p u b l ic a ti o n 1978.
V The in flu e n c e o f s c ro ta l warming on t e s t i c u l a r blood flow and endocrine fu n c tio n in the r a t . J . - E . Damber and P.O.
Janson: Acta p h y s io l, scand., accepted f o r p u b l ic a ti o n 1978.
oo.
CONTENTS
LIST OF PUBLICATIONS 2
ABBREVIATIONS 4
INTRODUCTION 5
AIM OF THE PRESENT STUDY 12
METHODOLOGICAL CONSIDERATIONS 13
Animals and a n a e s th e s ia ,13
T e s t i c u l a r blood flow measurements,13 D i r e c t measurement o f venous o u t f l o w , 13
I n d i r e c t measurement using Xenon-133 c l e a r a n c e , 13 I n d i r e c t measurement using r a d i o a c t iv e misrospheres, 15 Radioimmunoassay o f t e s t o s t e r o n e , ! 5
An tiserum ,15 S t e r o i d s , 15
Testosterone a s s a y ,16 S t a t i s t i c a l a n a ly s e s ,21
RESULTS AND COMMENTS 22
1. Methodlogical aspects o f t e s t i c u l a r blood flow measurements (Paper I ) , 22
2. The r e l a t i o n s h i p between t e s t i c u l a r blood flow and o utflow of tes to s tero n e in the spermatic vein (Paper I I ) , 24
3. The e f f e c t s o f LH and catecholamines on t e s t i c u l a r blood flo w and plasma te s to s te ro n e co n centra tion (Paper I I I ) , 24
4. T e s t i c u l a r blood fl ow and tes to s te ro n e conce ntra tions in the spermatic venous blood in r a t s with experimental cryptorchidism (Paper I V ) , 25
5. The i n flu e n c e o f s c ro ta l warming on t e s t i c u l a r blood flow and endocrine fu n c tio n (Paper V ) , 2 6
GENERAL DISCUSSION 27
SUMMARY AND CONCLUSIONS 35
ACKNOWLEDGEMENTS 38
REFERENCES 39
LIST OF ABBREVIATIONS
ABP androgen binding p ro te in
ACTH a d r e n o c o r tic o tr o p h ic hormone
cAMP ad en o sin e -(c y c lic )-3 ',5 -m o n o p h o s p h a te
BSA bovine serum albumin
FSH f o l l i c l e s ti m u l a ti n g hormone
HCG human ch o rion ic gonadotrophin
i .a . i n t r a - a r t e r i a l l y i . p . i n t r a - p e r i t o n e a l l y LH l u t e i n i z i n g hormone
PE polyethyle ne
oo_ oxygen p a r t i a l pressure r c o r r e l a t i o n c o e f f i c i e n t SEM standard e r r o r o f the mean
5.
INTRODUCTION
The general background to the present i n v e s t i g a t i o n was the d isco ve ry in t h i s l a b o r a to r y (Carstensen e t a l . , 1973) t h a t su rg ical stress can depress plasma te s to s te ro n e co nce ntra tion s in male p a tie n ts without accompanying changes in plasma LH- or FSH-concentrations. This dis co ve ry, to ge the r w ith s i m i l a r r e s u l ts re po rted in the l i t e r a t u r e , ( e . g . Aono e t a l . , 1976) i n i t i a t e d a search f o r o the r f a c t o r s , a p a r t from gonado
tr o p h i n s , i n flu e n c in g t e s t i c u l a r te s to s te r o n e production. One p a r t o f t h i s p r o j e c t was the study o f the p ossible r o l e o f t e s t i c u l a r blood flo w as a re g u l a to r y mechanism f o r t e s t i c u l a r endocrine fu n c tio n .
In the f i r s t p a r t o f t h i s se ctio n some general aspects o f the male gonad w i l l be discussed. Some poin ts regarding t e s t i c u l a r va scular s t r u c t u r e s , blood flo w and proposed vasc ular fu nc tio n s w i l l then be b r i e f l y reviewed.
General aspects o f t e s t i c u l a r physiology
I n flu e n c e o f p i t u i t a r y gonadotrophins: Male re prod u ctive a b i l i t y is dependent on the c a p a c ity of the t e s t i s to produce v i a b l e sperms and ade
quate q u a n t i t i e s o f androgens. These two main fu nctio n s o f the mammalian t e s t i s are known to be re gulated by gonadotrophic hormones produced by the p i t u i t a r y . Testosterone production takes place in the Leydig c e i l s o f the i n t e r s t i t i a l ti s s u e and is in flu en ced by LH. (Cooke e t a l . , 1972;
Eik-Ne s, 1975 ). The a c tio n o f LH is thought to be i n i t i a t e d by i n t e r a c t i o n w ith s p e c i f i c rece ptors loc ated on the Leydig c e l l membrane ( C a t t and Dufau, 19 76). The binding of LH to the re ce pto r leads to the a c t i v a t i o n o f aden ylate cy clas e, causing a s ti m u l a ti o n o f cAMP production and a c t i v a t i o n o f cAMP dependent p ro te in kinase ( f o r r e f . see Rommerts e t a l . ,
1974 ). I t has been shown t h a t a p o s i t i v e c o r r e l a t i o n e x i s t s between p ro te in kinase a c t i v a t i o n and s ti m u l a t i o n o f tes to s te ro n e production (Cooke e t a l . , 1976 ). The in flu e n c e o f FSH on ste ro idogenesis in the t e s t i s is u n c le a r.
For example, Odell and S w erdloff (1973) found t h a t FSH augmented t e s t o sterone production induced by LH in v i v o . On the o th er hand, Moger (1977) observed no e f f e c t on serum androgen co nce ntra tion s in r a t s a f t e r acute FSH tre a tm e n t, n e i t h e r did Heindel e t a l . (1975) f i n d any e f f e c t o f FSH on cAMP l e v e l s in i s o la t e d i n t e r s t i t i a l c e l l s , whereas LH induced a marked s t i m u l a t i o n . These discrepancies in r e s u l ts may be due to the im p u rit y o f a v a i l a b l e FSH p re p a r a tio n s . However, a t the presept tim e, the possible e f f e c t o f FSH on the Leydig c e l l remains an open question.
I t is more g e n e r a l ly agreed upon t h a t FSH evokes reponses in the seminiferous tubules where the spermatogenesis occurs. The S e r t o l i c e l l is considered to be the primary t a r g e t f o r FSH w it h i n the t e s t i s . A s p e c i f i c androgen binding p ro te in (ABP) is produced by the S e r t o l i c e l l s fo ll o w i n g FSH a d m i n is tr a tio n to immature hypophysectomized r a t s (Hanson e t a l . , 1974 ). I t has been suggested t h a t the fu n c tio n o f ABP is to cause an accumulation o f androgen w it h i n the seminiferous tu b u le s , thus m a in ta in in g an androgenic m i l ie u necessary f o r spermatogenesis. The mode o f ac tio n o f FSH on the S e r t o l i c e l l s is thought to be the a c t i v a t i o n of the adenylate-cyclase-cAMP system ( f o r r e f . see S tein b e rg er and S te in b e rg e r , 19 77 ).
I t has also been demonstrated t h a t FSH can s ti m u l a te the ar om atisa
ti o n o f te s to s te r o n e to e s t r a d i o l - 1 7 3 in c u ltu re d S e r t o l i c e l l s , and t h i s response to FSH could be simulated using dibutyryl-cAMP (Dorr in gton and Armstrong, 19 75). Since the tes to s te ro n e synthesis o f the Leydig c e l l s is i n h i b i t e d by e s t r a d i o l , i t has been suggested t h a t a sh o rt-lo o p control system e x i s t s where estrogens produced by the S e r t o l i c e l l s i n h i b i t stero id ogenesis in the Leydig c e l l s ( f o r r e f . see Connell and Connell, 19 77). The Leydig c e l l s also contain estrogen re ceptors (vanBeurden- Lamers e t a l . , 1974) and exogenous a d m in is tr a tio n o f estrogens to i n t a c t male r a ts have been re po rted to r e s u l t in a decrease o f te s to s te ro n e con
c e n tr a tio n s in plasma. This f a l l in te s to s te ro n e preceded the re duction o f LH ( e . g . Chowdhury e t a l . , 1974) i n d i c a t i n g a d i r e c t e f f e c t o f e s t r o gens on the Leydig c e l l s .
P r o l a c t in would appear to be another p i t u i t a r y f a c t o r o f importance f o r the t e s t i c u l a r fu n c tio n through i t s suggested a b i l i t y to incre ase the s e n s i t i v i t y o f the Leydig c e l l s to LH s ti m u l a ti o n (Bartke and D a l t e r i o , 19 76). I t is belie ve d to do t h i s by incre asin g the amount o f e s t e r i f i e d ch o le s te ro l a v a i l a b l e f o r conversion in to sex s te ro id s (B a rtk e , 19 69 ).
Thus, the c e n tr a l r o l e o f the p i t u i t a r y in the re g u l a t i o n o f the male gonad is i n d i s p u t a b l e , although the exac t mechanism o f a c tio n o f the gonadotrophins is f a r from being e x a c tly understood. Further research is necessary i f our knowledge in t h i s f i e l d o f male re prod u ctive physiology is to advance.
In flu e n c e o f nongonadotrophic f a c t o r s : The past years have re s u lte d in an incre asing number o f observations i n d i c a t i n g t h a t f a c t o r s othe r than gonadotrophins may play important r o le s in the re g u l a t i o n o f t e s t i c u l a r fu n c t io n , e . g . biogenic amines ( f o r r e f . see Urry and E l l i s , 1977, p ro sta
glandins ( e . g . Saksena e t a l . , 19 73 ), temperature ( f o r r e f . see VanDemark and Free, 1970) and d i f f e r e n t kinds o f str es s ( e . g . Sutton e t a l . , 1973;
7.
Kreutz e t a l . , 1972; Carstensen e t a l . , 19 73 ).
I t is obvious t h a t the blood flow to and from the te s te s is im
p o r ta n t f o r t e s t i c u l a r f u n c t io n , since i t is responsib le f o r supplying hormones, n u t r i e n t s and gases, and f o r removing the waste and se creto ry products from the t e s t e s . Eik-Nes (1 9 6 4 ), using an in v i t r o perfusion system, demonstrated t h a t the s e cretio n o f te s to s te ro n e v a ried d i r e c t l y w it h the r a t e o f p e rfu sio n. This fi n d i n g i n d ic a te s t h a t c i r c u l a t o r y v a r i a t i o n in the t e s t i s may be one o f the f a c t o r s determining the t e s t o sterone s e cretio n r a t e in v i v o . Since catecholamines and pro stag landin s ar e known to be v a s o a c tiv e , i t is pos sible t h a t these substances a f f e c t t e s t i c u l a r hormone se c re tio n p r i m a r i l y v ia an in flu e n c e on the va scular bed.
T e s t i c u l a r blood flow
The sc ro ta l t e s t i s is a r t e r i a l l y supplied by the i n t e r n a l spermatic a r t e r y o r i g i n a t i n g from the a o rta a t the le v e l o f the renal a r t e r y ( f o r r e f . see S e t c h e l l , 19 70 ). One o f the s t r i k i n g fe a tu r e s o f the va scular anatomy is the presence o f a la r g e number o f convolutions in the sperma
t i c a r t e r y w it h i n the pampiniform plexus. The t e s t i c u l a r veins j o i n the pampiniform plexus where they break up in to many f i n e ve in s , which are in close contact w it h the c o i l s o f the spermatic a r t e r y . The r o l e o f t h i s vasc ular o rg a n iz a tio n in the co u nte rcu rren t heat exchange has been under
stood f o r a long time ( e . g . Waites and Moule, 19 61 ). The a b i l i t y of the pampiniform plexus to exchange r e s p i r a t o r y gases has also been recognized
(Cross and S i l v e r , 19 62). More l a t e l y i t has been shown t h a t tes to s tero n e can be tr a n s f e r r e d from vein to a r t e r y in the pampiniform ple xus, which thus serves as a te s to s te r o n e co n ce ntra ting mechanism f o r the t e s t i s
(Free and J a f f e , 19 75 ). Furthermore, va so active substances in j e c t e d into the i n t e r n a l spermatic vein r e s u lte d in a marked reduction in l a t e r a l pressure and flow volume in the t e s t i s a r t e r y , w ith o ut changing c e n tr a l blood pressure , i n d i c a t i n g a passage o f these substances from vein to a r t e r y in the pampiniform plexus ( f o r r e f . see Free, 19 77 ). Within the t e s t i s , the a r t e r i e s f o ll o w d i f f e r e n t p attern s of c o i l i n g on the surfa ce and then descend i n to the i n t e r i o r p ortio n o f the t e s t i s , th ere d iv id i n g in to a r t e r i o l e s and c a p i l l a r i e s . The surfa ce convolutions o f the i n t r a - capsular p ortio n o f the t e s t i c u l a r a r t e r y may have a number o f phy siolo g ic a l i m p li c a t io n s , e . g . maintenance of optimum temperature in the t e s t i s , o b l i t e r a t i o n o f the p ulse, and reduction of blood flow ( S te i n b e r g e r , 1970 ).
19 70 ); i n t e r t u b u l a r c a p i l l a r i e s running p a r a l l e l to the seminiferous tubules near the Leydig c e l l s in the i n t e r s t i t i a l spaces, and p e r i tu b u l a r c a p i l l a r i e s which run a t r i g h t angles to the i n t e r t u b u l a r c a p i l l a r i e s around the i n d i v i d u a l tu b u le s .
Blood le a vin g the t e s t i s i s the major route f o r the tra n s p o rt o f androgens from the te s te s to the r e s t o f the body. However, the r e l a t i o n ship between t e s t i c u l a r blood flow and the se c r e tio n o f androgens is not f u l l y understood. I t i s pos sible t h a t the decrease in plasma t e s t o sterone brought about by p ro staglandins ( e . g . Saksena e t a l . , 1973) is mediated by a re duction in t e s t i c u l a r blood f l o w , since Free and T ils o n (1973) found a p a r a l l e l re du cti o n in plasma te s to s te r o n e co n centra ti on and t e s t i c u l a r blood flow a f t e r in fu sio n o f pro sta g lan din e E2 in r a t s . In the ovary, a tendency towards a p o s i t i v e r e l a t i o n s h i p between ovarian blood flow and progesterone s e c r e tio n r a t e has been re porte d (Romanoff, e t a l . , 1962). A s i m i l a r r e l a t i o n s h i p between blood flow and s te r o id s e c r e tio n has also been demonstrated f o r the adrenal gland ( f o r r e f . see Eik-Nes, 19 75 ).
Measurement o f t e s t i c u l a r blood flow
Many d i f f e r e n t techniques have been used to measure t e s t i c u l a r blood flow in d i f f e r e n t species ( f o r r e f . see S e t c h e l l , 19 70). The use o f i n e r t gas clearance r a t e , i n d i c a t o r f r a c t i o n a t i o n technique and f r i c t i o n f l o w meters has g r e a t l y enhanced our understanding o f t e s t i c u l a r blood flow and i t s co ntro l (Gomes and VanDemark, 19 74 ). Although many o f the tec h niques have y i e ld e d r e s u l t s o f approximately the same magnitude, very few systematic comparisons o f methods have been performed. For each technique c e r t a i n p r e r e q u i s it e s must be f u l f i l l e d . For example, the commonly used Xenon-clearance technique re q u ire s a homogeneous perfu sio n in o rd er to be v a l i d (K e ty, 1951) and so f a r i t is u nclear whether t h i s is the case f o r the t e s t i s . Larson and Foote (1974) found a s i g n i f i c a n t l y higher Krypton-85 cle ara nce from the c r a n i a l p a r t o f the r a b b i t t e s t i s as compared w ith the caudal p a r t suggesting an heterogeneous d i s t r i b u t i o n o f flow in the t e s t i s o f t h i s spec ies. The use o f i n d i c a t o r f r a c t i o n a t i o n techniques ( S a p i r s t e i n , 1958) would o f f e r some adva ntages,in comparison to othe r techniques, since they can be ap p lied to a s u r g i c a l l y i n t a c t organ, they r e q u ire no homogeneously perfused vascular bed, and they enable the measuring event o f several organ blood flows simultaneously.
9.
The technique o f S a p i r s t e i n may, however, y i e l d f a l s e l y low fl ow values in organs w ith a very high blood flo w r a t e due to an incomplete e x t r a c t i o n o f the i n d i c a t o r s used. In c o n tra s t to the i n d i c a t o r f r a c t i o n a t i o n technique using rubidium -86 or potassium-42 the use o f r a d i o a c t iv e micro
spheres has been shown to be s u i t a b l e f o r blood flow measurements also in hyperperfused tissu e s (Jansor. and A l b re c h t, 1975 ). The technique was modified f o r use in small l a b o r a to r y animals by Rudolph and Heymann
(1967) and was r e c e n t l y ap p lied to the r a t (Bruce, 1976 ).
Biogenic amines and t e s t i c u l a r blood flow
I t has been re ported by Levin e t a l . (1967) t h a t systemic a d m in is tra ti o n o f epinephrine to normal men w i l l decrease plasma tes to s tero n e con
c e n t r a t i o n . Since i t was known t h a t in fu s io n o f ep in e p h rin e, or norep ine- * p h r in e , in t o the spermatic a r t e r y o f rams led to a marked decrease in t e s t i c u l a r blood f l o w , i t was suggested t h a t t h i s e f f e c t on tes to s tero n e le v e l s was due to a catecholamine induced va s o c o n s tric tio n in the t e s t i s (S e tc h e ll e t a l . , 19 66). Also in r a t s , i t has been reporte d t h a t local a d m i n is tr a tio n o f catecholamines to the t e s t i s decreases blood flow
( J a f f e and Free, 1972). However, i t is not y e t c l e a r whether s y s te m ic a lly adm inistered catecholamines are able to decrease t e s t i c u l a r blood flow to such an e x te n t t h a t i t depresses the production o f androgens from the t e s t i s .
In c o n tra s t to the fi n d i n g s mentioned above, Eik-Nes (1969) reporte d a s ti m u l a ti o n of te s to s te r o n e production when d i f f e r e n t catecholamines were infused v ia the t e s t i c u l a r a r t e r y o f dogs under p e n to b a r b ita l anaesthe
s i a . The g r e a t e s t e f f e c t was obtained with isoprote re no l and t h i s s ti m u l a t i o n was suppressed by tre atm en t w it h an ad re nerg ic b e ta -r e c e p to r blocking agent. I t has also been shown t h a t epinephrin e stim u la te s the production o f cAMP from t e s t i c u l a r homogenate o f dogs and r a t s in v i t r o (Murad e t a l . , 1969). The c o n tr a d i c to r y experimental r e s u l t s obtained in studies o f the e f f e c t o f catecholamines on t e s t i c u l a r fu n c tio n may stem from the v a r i e t y o f e f f e c t s t h a t these substances have on both general and loc al c i r c u l a t i o n and metabolism. When discussing t e s t i c u l a r endocrine fu n c tio n the e f f e c t s o f biogenic amines on gonadotrophin re le a s e must also be con
sidered ( e . g . Ojeda and McCann, 1973).
T e s t i c u l a r fu n c tio n may be influ en ced by catecholamines reaching th e tes tes from c i r c u l a t i n g blood, or l o c a l l y releas ed a t nerve endings on,JD rin close p ro xim ity t o , vessels and Leydig c e l l s . Except in the case o f the t e s t i s o f the swan (Baumgarten and H o l s t e i n , 1 9 6 8 ), h i s t o l o g i c a l techniques have not i n d ic a te d t h a t t e s t i c u l a r nerve f i b e r s are associated with the Leydig c e l l s ( e . g . Norberg e t a l . , 1964; Stach, 19 62 ).
The nerves in the te s te s o f most species seem to be r e l a t e d to the blood vessels i n d i c a t i n g t h a t they have a r o l e in the r e g u l a t i o n o f blood flow (Elia sson and R i s l e y , 19 67 ). Simultaneous measurement o f t e s t i c u l a r blood flow and te s to s te r o n e production would appear to be one way o f overcoming the d i f f i c u l t i e s associated w ith studies o f the r o l e o f b io genic amines in the r e g u l a t i o n o f t e s t i c u l a r fu n c t io n .
The r o l e o f gonadotrophins in the re g u l a t i o n o f t e s t i c u l a r blood flow I t is well es ta b lis h e d t h a t LH causes a pronounced increase in ovarian blood flo w ( e . g . Janson, 19 75 ). So f a r , no c l e a r e f f e c t o f LH on t e s t i c u l a r blood flow has been re p o rte d .
Following a d m i n is tr a tio n o f HCG to r a t s , Hartman e t a l . (1950) found morphological signs o f a v a s o d i l a t a t i o n in the r a t t e s t i s . However, other authors have f a i l e d to demonstrate any e f f e c t o f gonadotrophins on t e s t i c u l a r blood flow ( f o r r e f . see S e t c h e l l , 19 70 ). On the o th e r hand, i t has been reported t h a t hypophysectomised r a t s have a decreased r e l a t i v e blood flow in the t e s t i s (S e tc h e ll e t a l . , 1969). Furthermore, in a study o f the development o f the t e s t i c u l a r va sc ulatu re in the r a t , Kormano
(1967) found a s i g n i f i c a n t developmental phase in the microvasculature between the ages o f 20 and 35 days, which may i n d i c a t e t h a t gonadotrophins p la y a r o l e in promoting va scular growth in the t e s t i s . In the r a b b i t , both the flow o f blood from the t e s t i s and the se c re tio n o f tes to s tero n e inc rease a f t e r mating ( f o r r e f . see Eik-N es, 1975 ). In the l i g h t o f these fin d in g s i t is d i f f i c u l t to e x p la in the r e p o r t o f Davidson e t a l . (1 9 7 4 ), which presents r e s u l t s i n d i c a t i n g a decrease in t e s t i c u l a r blood flo w 1 hour a f t e r i n j e c t i o n o f FSH i n to r a t t e s t i s t i s s u e .
The i n flu e n c e o f temperature and cr yptorchidism on t e s t i c u l a r blood flow and endocrine fu n c tio n
The impaired spermatogenesis in the heated or in tr a -a b d o m in a l l y r e tain ed t e s t i s is well documented ( f o r r e f . see VanDemark and Free, 19 70 ).
The c e l l s most s e n s i t iv e to heating are l a t e pachytene primary spermato
cytes and e a r l y round spermatids which show h i s t o l o g i c a l evidence of damage w it h i n 2 - 4 hours a f t e r heating (Chowdhury and S t e in b e rg e r , 19 70 ).
n .
The same c e l l s , and also the long spermatids, ar e damaged in the c ry p to rc h id t e s t i s ( e . g . Par vinen , 1973). I t is g e n e r a l ly agreed t h a t the d i f f e r e n c e in temperature between the scrotum and the i n t r a abdominal c a v i t y induces the impairment o f t e s t i c u l a r fu n c tio n seen in cryp torch idism . However, the exac t mechanism is s t i l l u nc le ar.
I t was re porte d by Glover (1965) t h a t the u n i l a t e r a l cryp to rch id r a t t e s t i s showed a s l i g h t l y increased r e l a t i v e blood flow a^s compared to the s c ro ta l t e s t i s . S i m i l a r observations o f flow have been made, by Ei k-Nes, on the c o n g e n i t a l l y c ryp to rch id dog (1 9 6 6 ). I t was suggested by the l a t t e r au th o r, t h a t a l t e r a t i o n o f blood flow from the c r yp torch id t e s t i s could be a compensatory mechanism to incre ase tes to s te ro n e se cre
t i o n . On the othe r hand, t e s t i c u l a r blood flow appears to be r e l a t i v e l y i n s e n s i t i v e to loc al warming up to abdominal tem pera tu re, a t l e a s t during a short term exposure ( e . g . Waites e t a l . , 19 73 ). Furthermore, using the i s o la t e d perfused t e s t i s o f the r a b b i t , Ewing and VanDemark (1963) re ported a decreased r a t e o f blood flow as a r e s u l t o f incre asing the temperature. I t was r e p o rte d , in the case o f the ram (Waites and S e t c h e l l , 19 64 ), t h a t the a p p l i c a t i o n o f s u f f i c i e n t heat to cause spermato- genic damage re s u l te d in hypoxia in the t e s t i s , but did not c o n s i s t e n t l y a l t e r the blood flo w . Thus, a pos sible explanatio n o f the impairment o f t e s t i c u l a r f u n c t io n , as seen in c ryp to rch id ism , or a f t e r lo c al heating o f the scrotum, may be a dis turbance in n u t r i t i o n and oxygenation, and t h e r e f o r e , v a r i a t i o n s in t e s t i c u l a r blood flow may be im porta nt.
The s e creto ry fu nc tio n s o f the Leydig and S e r t o l i c e l l s ar e e s s e n tia l f o r a normal spermatogenesis. There are r e s u l t s suggesting an impaired S e r t o l i c e l l fu n c tio n in experimental cr ypto rchidism in the r a t (Hagenäs and R i tz e n , 1976; Schenk and Neuman, 1977). Regarding the Leydig c e l l s , however, opinions d i f f e r as to whether these c e l l s are damaged or not.
Several re p o rts in the l i t e r a t u r e i n d i c a t e an impaired Leydig c e l l fu n c t io n , i n f e r r e d from reduced s te r o id pro duction, in the cry p to rc h id t e s t i s , ( f o r r e f . see VanDemark and Fre e, 19 70). On the othe r hand, some authors have shown a normal ( e . g . Ruokonen e t a l . , 1973) or even increased (Hagenäs 1977) s t e r o id content in the r e ta in e d t e s t i s in e x p e rim e n ta lly c r y p t o r chid r a t s . Although th e re is a r e p o r t o f normal s te r o id content in i n t e r s t i t i a l ti s s u e o f r a t t e s t i s a f t e r short term heat exposure ( C o l l i n s and Lacy, 1 9 67 ), info rm ation i s d e f i c i e n t where the Leydig c e l l fu n c tio n a t temperatures known to induce impairment o f spermatogenesis is con
cerned.
AIM OF THE PRESENT STUDY
A g re a t q u a n t i t y o f in fo rm ation regarding the male gonad has been obtained from experiments w ith r a t s . Furthermore, the l a b o r a to r y r a t has a continuous spermatogenesis and Leydig c e l l a c t i v i t y throughout the y e a r . This animal was th e r e f o r e se lecte d f o r a s e rie s o f experiments on the f u n c tio n a l r o l e o f t e s t i c u l a r blood flo w .
In the f i r s t p a r t o f the study some methodological aspects o f t e s t i c u l a r blood flow measurements were studied (Paper I ) . The second p a r t deals w ith the pos sible r e l a t i o n s h i p between t e s t i c u l a r blood flow and t e s t i c u l a r endocrine fu n c tio n and the hyp oth etica l involvement of hormones in the r e g u l a t i o n o f blood flo w in the t e s t i s (Papers I I and I I I ) .
Special a t t e n t i o n was paid to the p o s s i b i l i t y t h a t the e f f e c t o f c a t e cholamines on te s to s te r o n e production was brought about by a c tio n on t e s t i c u l a r v e ss els. The l a s t p a r t o f the study was performed in order to analyse the p ossible r o l e o f t e s t i c u l a r blood flo w and Leydig c e l l func
t i o n in promoting the d e l e t e r i o u s e f f e c t o f heat and cryptorchidism on t e s t i c u l a r fu n c tio n (Papers IV and V ).
Thus, the purposes o f the present study were:
1. to e s t a b l i s h a technique f o r measuring t e s t i c u l a r blood flow in the r a t using r a d i o a c t i v e microspheres, and to compare t h i s technique w ith the Xenon-133 cle ara nce technique and with the d i r e c t measure
ment o f spermatic venous o u tflo w .
2. to study the d i s t r i b u t i o n o f blood flow to various p arts o f the t e s t i s and to i n v e s t i g a t e whether a rte r io -v e n o u s shunts o f fu n c tio n a l importance e x i s t in the t e s t i s .
3. to study the r e l a t i o n s h i p between t e s t i c u l a r blood flow and the o u tflow o f te s to s te r o n e in the spermatic v e in .
4. to study the e f f e c t s o f LH and catecholamines on t e s t i c u l a r blood flow and plasma te s to s te r o n e co n c e n tra tio n .
5. to ev alu a te the s e c r e tio n o f te s to s te r o n e from the cryp to rch id t e s t i s by measuring t e s t i c u l a r blood fl ow and te s to s te r o n e conce ntra tions in the spermatic venous blood.
6. to study the e f f e c t o f lo c al heating on t e s t i c u l a r blood flo w . 7. to determine the s e n s i t i v i t y o f the Leydig c e l l s in vivo to LH
s ti m u l a ti o n a t supernormal t e s t i c u l a r tem peratures.
METHODOLOGICAL CONSIDERATIONS
1 3.
Animals and anaesthesia
Male Sprague-Dawley r a ts weighing 300 - 450 g, were used. They were kept in standardized environmental conditions (te mperature: 25° C, l i g h t 05:00 - 19:00 h ). Standard r a t p e l l e t s and water were always a v a i l a b l e . Most animals were an aesth etized w ith sodium pen tob arbi
tone (Nembutal, Abbott) giv en , in a dose o f 40 mg/kg i . p . , as a s in g le i n j e c t i o n . In experiments where blood flows were measured, the animals were kept supine and body temperature was maintained by a heating pad.
In some o f the experiments (Paper IV) newborn r a t s were operated w ith i n 20 hours o f d e l i v e r y , to induce u n i l a t e r a l cr yp torch idism (Bergh e t a l . , 1978 ). Blood pressure was continuously monitored using a Statham P 23 AC trans du ce r, connected to a Grass Model 7 Polygraph. The a r t e r i a l pressure was recorded d i r e c t l y as a damped signal of the pulse pressure.
T e s t i c u l a r blood flow measurements
The anatomy o f the blood vessels supplying and d ra in in g the t e s t i s is s c h e m a tic ally i l l u s t r a t e d in F ig . 1.
D i r e c t measurement o f venous o u tflo w . A f t e r laparotomy, the r i g h t spermatic a r t e r y and vein were exposed. The spermatic vein was cannulated, using a p olyethyle ne c a th e te r (P E -5 0 ), a t l e a s t 5 mm from i z s e n tr y in to the cavai ve in . Blood flow measurement was s t a r t e d 2 - 3 rrnn a f t e r cannu- l a t i o n . The c a t h e t e r , 3 - 4 cm in le n g th , was kept in a f i x e d p o s itio n w ith i t s open end a t the l e v e l o f the i n f e r i o r vena cava. The spermatic venous blood was c o l le c t e d in preweighed glass tubes, during one-minute, a f t e r which the t e s t i c u l a r a r t e r y was l i g a t e d through a p re v io u s ly made midsc rotal in c is io n ( f o r d e t a i l s see Paper I ) . Immediately fo llo w in g the l i g a t i o n , the spermatic venous o utflow was measured f o r another one- minute p erio d. The blood flow to the t e s t i s was then c a l c u la te d as fo llo w s : T e s t i c u l a r blood flow = blood flow in the spermatic vein before l i g a t i o n o f the t e s t i c u l a r a r t e r y - blood flow in the spermatic vein a f t e r l i g a t i o n .
I n d i r e c t measurement using Xenon-133 c le a r a n c e . Xenon-133 dissolved in s t e r i l e s a l in e was obtained commercially (AB Atomenergi, Stu dsvik, Sweden).Approximately 2G - 40 yCi in 30 - 40 yl o f s a l in e was i n j e c te d percutaneously in to the t e s t i s w ith a g a s ti g h t Hamilton sy ringe. A Na-I c r y s t a l d e te c to r (Friesecke-Hoepfner 421 A) was used with a 10 mm e x i t diameter col 1 imat o r (Friesecke-Hoepfner 417 B), the dis tance between the c r y s t a l and the t e s t i c u l a r surfa ce being approximately 20 cm. The d e te c to r was connected to a Friesecke-Hoepfner 49 A sc a le r and a H e w le tt- Packard 7172 A s t r i p c h a r t re cord er was used f o r re co rd ing . A f t e r
background s u b tr a c tio n , recordings were p lo tte d on se m i-lo g a rit h m ic
paper, t i / 2 was c a lc u la te d from the washout curve and introduced in to the fo llo w in g fo rm u la, as given by Kety ( 1 9 5 1 ) , in order to obta in t e s t i c u l a r blood flow :
F = In 2 X K X 100
1 1 / 2
The p a r t i t i o n c o e f f i c i e n t (K) used was 0.85 (S e tc h e ll e t a l . , 1966 ).
Internal
spermatic artery and vein
Plexus pampiniformis
Testicular artery
Deferential artery
Ductus deferens
Fig . 1 . Schematic i l l u s t r a t i o n o f the vessels o f the r a t t e s t i s and ep ididymis.
1 5.
I n d i r e c t measurement using r a d i o a c t iv e microspheres. Radioac tive
"carbonized" microspheres w it h a diameter o f 15 ± 5 ym (range) were supplied by 3 M Co., St Paul, Minn. USA. They were suspended in 20%
D
dextran and a drop o f d eterge nt (Teepol ) was added to the so lu tio n to i n h i b i t ag gre gation. The r i g h t c a r o t id a r t e r y was cannulated and the t i p o f a PE-50 c a t h e t e r was introduced i n to the l e f t v e n t r i c l e o f the h e a r t. During the development o f the method, ca nnulation o f the l e f t c a r o tid a r t e r y was attempted in ord er to reach the l e f t v e n t r i c l e . I t was found t h a t r i g h t c a r o t id cannula tio n provided an e a s i e r ro u te.
However, in Papers I and I I , som experiments were performed w ith l e f t c a r o t id ca n n u la tio n . The p o s itio n o f the c a t h e t e r was checked a t the end o f some experiments by d i s s e c t i o n . About 50000 spheres were tr a n s f e r r e d to a glass chamber w ith a volume o f 0 . 9 ml. The design o f the chamber, and i t s f u n c t io n , a r e described in d e t a i l by Rudolph and Heyman (1 9 6 7 ).
The chamber co ntaining the sphere suspension was v ig orou sly a g i ta te d w it h a high frequency mechanical s t i r r e r , and the suspension was flushed
in to the heart f o r 30 seconds w it h 1 ml 0.15 M NaCl. From f i f t e e n seconds before u n t i l 15 seconds a f t e r the in fu s io n o f microspheres, blood was continuously withdrawn, a t a constant r a t e , from the t a i l a r t e r y ( " r e f e rence sample"). In each case, the blood was withdrawn v i a a PE-90 c a th e t e r i n to a disposable 2 ml p l a s t i c sy rin ge attached to a pump adapted f o r a s p i r a t i o n . At the end o f the experim ent, the r a t was k i l l e d by an overdose o f sodium pentobarbitone and d is se cted . R a d i o a c t i v i t i e s o f the
"re feren ce sample", the te s te s and some o th e r organs and tissu e s were measured in an automatic w ell s c i n t i l l a t i o n counter (Packard Auto-Gamma).
Each sample was counted f o r 3 minutes. By counting standards conta ining known numbers o f microspheres, from the ac tu al batches, the numbers o f spheres in d i f f e r e n t organs and tissu e s could be c a l c u la t e d . F ig . 2 i l l u s t r a t e s how regio nal blood flo w d i s t r i b u t i o n was c a l c u la t e d .
Radioimmunoassay o f tes to s tero n e
Anti serum. The te s to s te r o n e a n t i serum was a g i f t from Dr L. Edqvist a t the Royal V e te r i n a r y C o lle g e , Stockholm. I t was ra is ed ag ain st t e s t o - sterone-3oxime-BSA. The percentage c r o s s -r e a c tio n to 5 a - d i h y d r o t e s t o sterone was c a lc u la te d as described by Abraham (1969) and was found to be 40%.
S t e r o i d s . ( 1 , 2, 6, 7 - 3H )-T es to s tero n e was p u r i f i e d in the l a b o r a to r y by paper chromatography (Bush, 1961) and used f o r both assay and d eterm ina ti on o f recovery . Testosterone was dissolved in absolute ethanol to giv e a standard s o l u ti o n .
F ig . Z . Schematic o u t l i n e o f the p rep aratio n and c a l c u la t io n s o f t e s t i c u l a r blood flow using the "re ference sample" m o d ifi c a tio n o f the r a d i o a c t iv e microsphere technique.
Testosterone assa y. Two te s to s te r o n e radioimmunoassays were set up:
one, more s e n s i t i v e , method f o r the d eterm ina tion o f te s to s te r o n e con
c e n t r a t i o n in " p e r ip h e r a l" plasma ("pg-method") and another, less se ns i
t i v e , method f o r measurement o f tes to s tero n e in spermatic vein plasma and t e s t i c u l a r homogenate ( "ng-method"). The reason f o r using a less s e n s i t i v e method in these l a t e r samples, w ith expected high tes to s tero n e co n c e n tra tio n s , was to avoid e r ro rs due to extensive d i l u t i o n .
"Pg-method". One hundred pi o f plasma were e x tra c te d 3 times w ith 5 ml d ie th y l e th e r . Testosterone antiserum was d i l u t e d (1:1 50 000) in borate b u f f e r ( 0 .0 6 M, pH 8 . 0 ) co ntaining human albumin ( 1 . 5 mg/ml), human gammaglobulin (C.5 mg/ml) and 3H4-te s t o s t e r o n e (75 000 dpm/ml).
Two hundred pi o f the antibody so lu tio n were added to the d ried d ie th y l e th e r f r a c t i o n and incubated a t + 4° C o v e r n ig h t. Standard samples of te s to s te r o n e , ranging from 0 to 300 pg, were assayed in d u p l ic a te t o geth er w ith the experimental samples. A f t e r incubation w ith the a n t i
Bild borttagen – se tryckt version Image removed – see printed version
1 7.
body s o l u t i o n , 200 yl o f saturated ammonium s u l f a t e was added in ord er to se parate bound and unbound s te r o id by p r e c i p i t a t i o n o f the p ro te in f r a c t i o n . 200 yl o f each sup ernatant was then pipeted in to in d i v i d u a l polyethyle ne v i a l s , 10 ml o f a s o lu tio n co ntaining 27 .5 g Permablend (Packard, S c i n t i l l a t i o n Grade) dissolved in 5 1 toluene was added to each v i a l and counted in a l i q u i d s c i n t i l l a t i o n cou nter. Testoste rone co n centration in the unknown samples was c a lc u la te d using a computer programme based on the " l o g i t - l o g " method (Rodbard and Lewald, 1970) f o r the tra n s fo rm a tio n o f the standard curve. A t y p i c a l standard curve is shown in F ig . 3. The concentra tions o f tes to s te ro n e obtained from the standard curve were corr ected f o r blank values and e x t r a c t i o n a l losses.
The s e n s i t i v i t y o f the standard curve was 10 pg when applying the 95%
confidence l i m i t . The c o e f f i c i e n t o f v a r i a t i o n f o r between assay d u p l i cates was 11.5%. The mean re covery f o r the e x t r a c t i o n o f te s to s te r o n e was 91 .9 ± 0.5% and the mean value f o r water blanks was 8 . 0 ± 1.1 pg.
The i n flu e n c e o f c r o s s -r e a c tin g s te ro id s was te s te d by measuring the plasma te s to s te r o n e concentra tions in the same plasma samples w ith and w it h o ut a paper chromatographic step (Carstensen and Bäckström, 1976). The r e s u l t s are shown in Table 1. There was no s i g n i f i c a n t d if f e r e n c e in plasma te s to s te ro n e co n centra ti on between the two cases.
4 - 1
3- o
GO
O)o 1 -
>1II
0 -
-1 - 1
r T T T 1
I I--- 1--- 1--- 1--- 1
10 25 50 100 200
x = log unlabelled Testosterone, pg
Fig . 3 . Standard curve from a te s to s te ro n e assay expressed in l o g i t - log c o -o rd in a te s . The slope o f th e curve is - 2 . 7 9 .
"Ng-method". This method was used f o r the dete rm ina ti on o f t e s t o sterone in spermatic ve in plasma and t e s t i c u l a r homogenate. F i f t y yl o f spermatic vein plasma was e x tra c te d 3 times with 5 ml d i e t h y l e t h e r . From each t e s t i c u l a r homogenate» a sample corresponding to 0.1 g o f ti s s u e was ex tra c te d 3 times w ith chloroform . The combined chloroform e x tr a c t s were washed w it h 1 ml 0.1 M NaOH, then twice with 2 ml d i s t i l l e d w ater. The te s to s te r o n e antiserum was d i l u t e d 1:30 000 in borate b u f f e r ( 0 .0 5 M, pH 8 . 0 ) co ntaining g e l a t i n e (1 mg/ml), human gamma g lo b u lin (0.1 mg/ml) and ^ - t e s t o s t e r o n e (290 000 dpm/ml ) . The a n t i serum was added to the d ie t h y l e th e r f r a c t i o n , or al t e r n a t i v e ly to the chloroform f r a c t i o n , a f t e r evaporation o f the s o lv e n t. The samples were incubated f o r 3 h a t 20° C. Blank and standard samples o f te s to s te ro n e were assayed in d u p l ic a te to gether w ith the experimental samples. Separa
ti o n o f bound from unbound s te r o id and l i q u i d s c i n t i l l a t i o n counting were performed as described above in the “pg-method". The standard curve was f i t t e d to an equation given by Ledercq e t a l . (1 9 7 1 ). An example o f a standard curve i s shown in F ig . 4. The recovery f o r the e x t r a c t i o n o f te s to s te ro n e was 92.1 ± 0.84% and the c o e f f i c i e n t o f v a r i a t i o n f o r between-assay d u p lic a te s was 5.7%. Water blank values were close to zero. The s e n s i t i v i t y o f the method was defined as the sm allest d e te c ta b le amount o f te s to s te r o n e added to pooled plasma samples. I t was found to be 0 .2 n g /m l. A comparison between the r e s u l ts obtained a f t e r analyzin g samples w ith and w it h o u t chromatographic se para tion is shown in Table 1. No s i g n i f i c a n t d if f e r e n c e was found.
Table 1 Radioimmunoassay f o r tes to s tero n e in p e rip h e ra l plasma, spermatic vein plasma and t e s t i c u l a r homogenate o f r a ts w ith and with o ut paper chromatography.
Testosterone
No Chromatographed Unchromatographed
P eripheral plasma
(ng/ml) 11 4 .5 ± 2 . 0 4 . 2 ± 2.2
Spermatic vein
plasma (ng/ml) 6 51.3 ± 3.2 54.8 ± 3.7
T e s t i c u l a r homo
genate (ng/g) 6 155.2 ± 2 5 .0 121.8 ± 9.8
Values expressed as mean ± S.E.M.
19.
B / Bq
100
9 0-
8 0-
7 0-
6 0-
5 0-
4 0-
3 0-
0.5 2.5
Testosterone, ng
Fig. 4 . Standard curve from a tes to s te ro n e assay f i t t e d to a p ara b o l i c equation between 0 . 3 and 3 ng as given by Leclercq e t al . (1 9 7 1 ).
The v a l i d i t y o f the two te s to s te ro n e radioimmunoassays was also tes ted by adding known amounts o f tes to s tero n e to c h a r c o a l - t r e a t e d human female plasma. As seen in Table 2 the r e l a t i o n s h i p between added and recovered s te r o id was s a t i s f a c t o r y .
Fig . 5. i l l u s t r a t e s the plasma te s to s te ro n e co nce ntra tion s a t
d i f f e r e n t times a f t e r the induction o f b a r b i t u r a t e anaesthesia in 5 r a t s . No experiment in the present i n v e s t i g a t i o n exceeded 60 minutes, and during t h a t time the plasma te s to s te ro n e appeared to be s t a b l e .
Table 2 R e la tio n s h ip between the amount o f te s to s te r o n e added to c h a r c o a l - t r e a t e d female human plasma and t h a t measured by RIA.
Method Amount o f t e s t o - Amount o f t e s t o
sterone added sterone measured
50 57 .5
Pg-method 100 91 .3
(pg ) 150 135.6
0.75 0.74
Ng-method 1 0.97
(ng) 1.5 1.36
D i f f e r e n t amounts o f u n la b e lle d s te ro id s were dissolved in 0.1 ml plasma. Each value is the mean o f f i v e d eterm ina tion s.
y \
E .o.
o>
c Q)
Co 4^O)0)
4-1o (0
105 120 90
0 15 30 45 60 75
Time, minutes
F ig . 5. Plasma te s to s te r o n e concentra tions in in d i v i d u a l ra ts at d i f f e r e n t times a f t e r ind u ction o f b a r b i t u r a t e an esth esia. Each l i n e represents one a n im a l.
