Nagoya J. Med. Sci. 54. 00 - 00, 1992

THE EFFECT OF HYPOPHYSECTOMY
ON PROLUMINAL MOVEMENT OF
3H-ANDROGENS ACROSS THE
EPIDIDYMAL EPITHELIUM IN THE RATMASANORI YAMAMOTO, YOSH1KAZU TSUJI, TATSUYA NAGAI
and KOJI MIYAKE
Department of Urology, Nagoya University School of Medicine, Nagoya 466, Japan

ABSTRACTThe effect of hypophysectomy on transepithelial movement of 'H-androgen in the rat epididymis was
examined by using in vivo micropcrifusion of 3H-tcstosterone followed by in vivo micropuncture to obtain
peritubular and intraluminal fluids. Experiments were performed on aminals without hypophysectomy or on
aminals 5-6 days after hypophysectomy and 9-10 days after hypophysectomy. Tubules were peri fused with
Minimum Essential Medium containing 3H-testosterone. 14C-polyethyleneglycol was included in the perifusion fluid as a marker for contamination of the intraluminal fluid by peritubular fluid. Radioactivity of isotopes in the interstitial and intraluminal fluid was determined at I and 2 hours after perifusion and the percentage of peritubular isotopes appearing in the intraluminal fluid was determined. A sperm concentration
microassay was performed on micropuncture samples from the epididymal tubules to assess testicular contribution to the lumen content. Proluminal movement of'H-androgcn and intratubular sperm concentrationsin the caput epididymal tubules of rats 9-10 days after hypophysectomy were significantly decreased. Pruluminal movement of'H-androgen and intratubular sperm concentrations in the cauda epididymal tubules ofrats 9-10 days after hypophysectomy were significantly increased. These results suggest that proluminal androgen movement is controlled by the presence of some testicular product in the epididymal lumen.
Key Words: Testosterone, Epididymis, Hypophysectomy, Micropuncture, Mieruperifusion.

INTRODUCTION
The epididymis serves as a conduit and storage depot for spermatozoa, but beyond that theorgan maintains sperm maturation processes that result in the sperm cell's obtaining progressive
motility and fertility. Many of the details for the mechanisms underlying these biologically significant functions remain to be elucidated; however, it is now clear that the physiological funcitonsof the epididymis are very complex.
Although the mechanisms by which the epididymis performs this function of sperm maturation are not fully understood, the general consensus is that this process is affected by the fluid
milieu within the epididymal lumen and by the epithelial cells which produce it. It has long been
accepted that the epididymis is an androgen target tissue, that epididymal metabolism and epididymal epithelial secretion depend on the presence of androgens, and that, consequently, epididymal sperm maturation does not occur in the absence of androgens. 1.2.3) However, there is little
determination about local mechanisms that regulate androgen availability to either that basal orapical aspects of their respective epithelia.

Received for Publication in August 7, 1991

67

68
Masanori Yamamoto et al.

Recently, by using in vivo microperifusion followed by in vivo micropuncture we have shownthat transepithelial movement of 3H-androgen is against a concentration gradient. 4,5) It has been
demonstrated that follicle-stimulating hormone (FSH) can stimulate an increase of androgenconcentration in the caput epididymis,6) It is known that FSH stimulated the secretion of androgen binding protein (ABP) by Sertoli cells7) and that ABP is concentrated in the epididymal
lumen. 8) Thus, we have hypothesized that ABP is important in regulating intraepididymal androgen concentrations, These facts have also led us to investigate the effect of hypophysectomy
on transepithelial movement of 3H-androgen into epididymal tubules,

MATERIALS AND METHODSIn Vivo Perifusion of Epididymal Tract and Subsequent Micropuncture
Adult male Sprague-Dawley rats (450-670 g) were maintained in a constant temperature(25'C), constant humidity (50%) vivarium on a 12L: 12D cycle. Food and water were available
ad libitum. Animals were anesthetized and prepared for microperifusion and micropuncture of
the epididymal tubules as previously described,4,5,9) Briefly, a 100-flm-tip micropipette wasplaced in a micromanipulator and connected to a 3-ml glass syringe with PE-60 tubing. The pipette, tubing, and syringe were filled with perifusion fluid. The perifusion fluid was 3 ml of lissamine-green-dyed Minimum Essential Medium (pH 7.0-7.4; Gibco Laboratories, Grand Island, NY) containing 80-flCi 3H-Testosterone (specific activity 55.2 CilmM; New England Nuclear, Boston, MA) and 4.0-flCi 14C-polyethyleneglycol C4C-PEG, specific activity 15.0 mCilg,
New England Nuclear). The filled syringe was attached to a perifusion pump (model 341B, Sage
Instruments, Cambridge, MA). Priming and sustaining perifusion rates for the caput and cauda
epididymal tubules were performed as previously described. 4) The micropipette was inserted directly through the caput epididymal tunica, and the pipette tip was left in the interstitial space as
described previously.4) Samples of fluid from the interstitial space and intraluminal fluids of thetubule were collected by micropuncture at one and two hours after initiation of the sustaining
perifusion. After the samples were collected, cell-free fluids were obtained by centrifugation anddivided into three aliquots which were transferred into vials containing 3 ml of scintillation fluid,and 3H- and 14C-radioactivity was determined by scintillation spectrophotometry.
Since 14C-PEG is excluded by the blood epididymal barrier, it was included in the perifusion
fluid as a marker for contamination of the intraluminal fluid by blood or peritubular fluid. 4) All
intraluminal 3H-androgen data were corrected for contamination following 14C-PEG. Radioactivity in the tubule lumina was expressed as the percentage of radioactivity appearing in thesame volume of interstitial fluid surrounding the same tubule at that same time period (intraluminal fluid counts per minute [CPM] divided by interstitial fluid CPM x 100).
Intratubular Sperm Concentrations
Sperm concentrations in micropunture samples of fluid from the caput and cauda epididymis
were determined with a sperm concentration microassay. Briefly, spermatozoa were obtained byin vivo micropuncture, put through a microdilution procedure with 1% hyaluronidase, andcounted on a hemocytometer ( x 3) using standard techniques.
Treatment Groups
In addition to a group of nine intact, untreated control rats there were two groups as described below.
1) Five-six day hypophysectomy (5-6d Hypox) group: Animlas were hypophysectomized byentering hypophyseal fossa through a cervical incision on Day O. These animals were maintainedon water containing 1% sodium chloride and 5% dextrose. On Day 5 or 6, they were subjected

69
PROLUMINAL MOVEMENT OF 3H-ANDROGEN

to studies as described above of proluminal 3H-androgen movement into the caput and cauda
epididymidis and of sperm concentrations in the caput and cauda lumina.
2) Nine-ten day hypophysectomy (9-lOd Hypox) group: Animals were hypophysectomized
on Day O. On Day 9 or 10, they were subjected to the same studies as the 5-6d Hypox animals.
After completion of each experiment, the hypophyseal fossa was examined for presence of
pituitary tissue. Animals with residual pituitary tissue were excluded from the study.
Data Analysis
Chauvenet's criterion was applied to all the data. IO ) The data are presented as mean andSEM. All multiple comparisons were made by the Kruscal Wallis test!!) for nonparametric data
followed by the Wilcoxon rank sum test (p < 0.05).

100% 14 C- PEG Remaining75(%) 5025o100% 3H - Androgen Remoining
0 1 hour Perifusion_
2 hour Perifusion

75(%) 5025oFig. 1.

Control

5-6 d9-10 d

Hypox

Hypox

Characterization of radiolabeled compounds in peritubular fluid from caput
epididymal interstitial space in control animals and those hypophysectomized after one and two-hour perifusion. Values were expressed as apercentage of original perifusion fluid 14C-PEG or 3H-androgen concentration remaining in the fluid from the peritubular space at either one or twohours after perifusion. No significant differences in isotope concentrations
remaining around the caput tubules were found between groups or times.

70
Masanori Yamamoto et al.

RESULTS
Proluminal movement of.1H-androgen and intraluminal sperm concentrations in the caput epididymal tubules
Isotope concentrations remaining in the perifusion fluid around the caput tubules after one
and two hours were not significantly different whether originating from 14C-PEG or 3H-T
(Figure 1). This was true within the control and within both hypophysectomized groups. Additionally, there were no significant differences in the isotope concentrations between experiments(Fig. 1). These data demonstrate that the radiolabeled compounds available to the tubules wererelatively similar from group to group and hour to hour.
After one and two hours perifusion of the caput epididymal tubules of the control rats,
315.3 ± 74.5% and 275.8 ± 51.5% of peritubular 3H-androgen concentrations, respectively, appeared in the intraluminal fluid (Fig. 1). These values were not altered in the 5-6d Hypox group,
but the 9-lOd Hypox animals demonstrated a significantly reduced net proluminal movement of
3H-androgen (91.5 ± 39.1 % and 44.44 ± 9.1 % at one and two hours, respectively), and thecaput tubules lost their capacity to move 3H-androgen against a concentration gradient (Fig.2).

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5-6 d

9-10 d

Hypox

Hypox

Perifusion

2 hours Perifusion

Proluminal movement of JH-androgen into the tubules of the caput epididymidis in control animals, those 5 or 6 days after hypophysectomy (5-6d
Hypax) and those 9 or 10 days after hypophysectomy (9-lOd Hypox). Asterisk shows significant difference (p < 0.05).

71
PROLUM1NAL MOVEMENT OF 'H-ANDROGEN

Intraluminal sperm concentrations in the control caput tubules were 0.72 ± 0.07 x 10 9 sperm/
m!. These values were not significantly affected in the 5-6d Hypax group, but they were significantly reduced int eh 9-lOd Hypox group (Fig. 3).

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Fig. 3.

5-6 d
Hypox

9-10 dHypox

1ntratubular sperm concentrations of lumen fluids in the caput epididymidisof control group, 5-6d Hypox group and 9-lOd Hypox group. Treatmentgroup data are compared statistically with the control data. Asterisk shows
significant difference (p < 0.05).

Proluminal movement of 3 H-androgen and intraluminal sperm concentrations in the cauda epididymal tubules
Isotope concentrations remaining around the cauda tubules after one and two hours perifusian were stable in all experimental groups (FigA). After one and two hours perifusion of thecauda epididymal tubules of the control rats approximately 120% of peritubular 3H-androgen

72

Masanori Yamamoto et al.

concentrations appeared in the intraluminal fluid (Fig.5). These values were significantly increased in both the 5-6d and 9-lOd Hypox groups, but the one hour value in the 5-6d Hypox
group was not significantly different from that of the control group (Fig.5). Intraluminal sperm
concentrations in the control cauda tubules were 1.85 + 0.045 x 10 9 sperm/ml. These values
were significantly increased in both 5-6d and 9-lOd Hypox groups (Fig.6).

100% 14C- PEG Remaining75(%) 5025

0...0..-.......100% 3H- Androgen Remaining

CJ 1 hour Perifusion

75_

2 hours Perifusion(%) 5025

0.4.---'--

Control

Fig. 4.

5-6 d

9-10 d

Hypox

Hypox

Characterization of radiolabeled compounds in peritubular fluid from
cauda epididymal interstitial space in control animals and those hypophysectomized after one and two-hour perifusion. Values were expressedas the percentage of original perifusion fluid 14C-PEG or 3H-androgen concentration remaining in the fluid from the peritubular space at either one or
two hours after perifusion. Isotope concentrations remaining around thecauda tubules after one and two-hour perifusion were stable in all experimental groups.

73
PROLUMINAL MOVEMENT OF 3H-ANDROGEN

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500

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5-6 d

9-10 d

Hypox

Hypox

Perifusion_Zhou,s Peri fusion

Fig. 5.

Proluminal movement of 3H-androgen into the tubules of the cauda epididymidis in control animals, those 5 or 6 days after hypophysectomy (5-6d
Hypox) and those 9 or 10 days after hypophysectomy (9-lOd Hypox.). Asterisk shows significant difference (p < 0.05).

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5-6 d9-10 d

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Hypox

Intratubular sperm concentrations of lumen fluids in the cauda epididymidis of the control group, 5-6d Hypox group and 9-10d Hypox group.
Treatment group data are compared statistically with the control data. Asterisk shows significant difference (p < 0.05).

74
Masanori Yamamoto et ai.

DISCUSSION
Antigrade, transepithelial 3H-androgen movement was maintained in the caput epididymis at
5 to 6 days but not at 9 to 10 days after hypophysectomy (Fig. 1). Likewise, intraluminal caput
sperm concentrations were still normal in the 5-6d Hypox group, but were significantly decreased in the 9-lOd Hypox rats (Fig.3). These data suggest that a period of ten days after hypophysectomy is sufficient time for clearance of pre-hypophysectomy testicular contribution fromthe caput epididymal lumen, and that there is a loss of antigrade proluminal androgen movement coincident with the loss of testicular contribution. It has been demonstrated that treatmentof hypophysectomized rats with FSH can stimulate an incrase of androgen concentration in thecaput epididymal tissues greater than that seen with luteinizing hormone (LH) alone. 6 ) FSH
stimulates the secretion of ABP by Sertoli cells 71, and ABP is concentrated in the epididymal
lumen. 8) Thus, we have hypothesized that some testicular product in the epididymal lumen, most
likely ABP, is needed to maintain normal proluminal androgen movement.
After one and two-hour perifusion of the cauda epididymal tubules of control rats approximately 120% of peritubular 3H-androgen concentrations appeared in the intraluminal fluid(Fig.5). These values were significantly increased in both the 5-6d and 9-lOd Hypox groups(Fig.5). It is speculated that if the epididymal flow rate was decreased in the Hypox rats, butsome epididyaml absorption continued, and increase in intraluminal concentrations of testicular
product resulting in an increase in proluminal androgen movement might occur. Increase in intraluminal sperm concentrations in the cauda tubules in both the 5-6d and 9-lOd Hypox groups
may be associated with increase of testicular protein and hormone. Although intraluminal fluid
volumes have been insufficient to alIow analysis of intraluminal ABP in any of the hypophysectomized groups, these results are consistent with the speculation that intraluminal ABP is an important factor in regulating transepithelial androgen movement in the epididymis.
ABP concentrations are high in rat caput epididymal tissue extracts and intraluminal fluids,
and these values decline in the more distal epididymis. 12,13,14) Attramadal et al. IS), Pelliniemi etal. 16 ), and Feldman l7 independently demonstrated that ABP is reabsorbed by the epithelial celIsof the rat initial segment and caput epididymis, and Gerrard et al. 18) have recently provided evidence that the ABP internalized by receptor-mediated endocytosis still retains its androgen. This
was the first direct evidence that ABP and adgrogens are internalized together in the caput
epididymal epithelium. Turner et al. 8) showed that large amounts of ABP are reabsorbed between the rete testis and caput epididymis. The intraluminal ABP: androgen ratio remains approximately 1: 1 from the proximal to the distal epididymis8), and this result is consistent with
our speculation that ABP sets the intraluminal androgen concentration.
Earlier reports I9 ,20,21) have indicated that vascular androgens alone are not sufficient to maintain the caput epithelial function, and that some other intraluminal, nonsteroidal testicular factor, is required. That this factor is ABP must be considered, especialIy since Anthony et al. 22,23)
found that epididymal ABP concentrations were significantly correlated with sperm fertility,
presumably through a role in affecting epididymal epithelial function.
Net proluminal movement of 3H-androgens across the tubule epithelia in the in vivo perifusion studies occurs in the order of caput, corpus epithelium > cauda epithelium > seminiferous
epithelium. 4,5) Intraluminal, radioimmunoassayable ABP concentrations follow a similar pattern. 8) This apparent association between intraluminal ABP and proluminal 3H-androgen movement and the absence of evidence of intraluminal androgen binding in either sperm celIs or intraluminal fluid provide evidence for the speculation that intraluminal ABP is the responsible
molecule for the proluminal, antigrade, transepithelial movement of 3H-androgens, and therefore, for the androgen microenvironment in the epididymis. Subsequent experiments will

75
PROLUMINAL MOVEMENT OF 3H-ANDROGEN

determine whether proluminal movement of 3H-androgen across the caput epididymal epithelium is returned to normal by supplementation with FSH or LH and whether proluminal movement occurs in the absence of spermatozoa.

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