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Original Articles |
Prince Henry’s Institute of Medical Research (Y.Z., R.I.M.) and the Institute of Reproduction and Development (N.G.W., D.M.d.K.), Monash Medical Center, and the Department of Anatomy, Monash University (N.G.W.), Clayton, Victoria 3168; and the Department of Surgery, Monash University, Inner and Eastern Health Care Group, Alfred Hospital (P.R.), Prahran 3181, Australia
Address all correspondence and requests for reprints to: Dr. R. I. McLachlan, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: Rob.MacLachlan{at}med.monash.edu.au
Abstract
Testosterone (T) treatment suppresses gonadotropin levels in normal men
and is a promising reversible contraceptive that induces azoospermia in
approximately 70% of subjects and oligospermia in the remainder;
however, the basis of this variable response is unclear. This study
aimed to investigate this reported variable response by examining the
spermatogenic process and quantitating germ cell number in men after
T-induced gonadotropin withdrawal. Ten normal fertile men (31–46 yr),
already planning to undergo vasectomy, either received T enanthate (200
mg, im, weekly) for 19–24 weeks (n = 5; TE group) or proceeded
directly to surgery (n = 5; controls), at which time a unilateral
testicular biopsy was taken, and germ cell numbers were estimated using
the optical disector stereological method. In response to TE treatment,
serum T levels rose 2-fold, and FSH/LH levels became undetectable.
Sperm counts fell to azoospermia in 4 men and to 21 million/mL in the
fifth man. The mean number of type A spermatogonia per 100 Sertoli
cells was unchanged, but type B spermatogonia fell markedly to 10% of
the control values, and later germ cell types decreased to 11–18% of
the control values. The pattern of germ cell suppression varied widely
and showed no relationship with sperm count or the time to azoospermia.
Despite the presence of elongated spermatids (1.4–20% of the
control), four men remained azoospermic. Two TE subjects with similar
early germ cell complements and elongated spermatid numbers had sperm
counts of zero and 21 million/mL; the latter man demonstrated marked
variability in germ cell numbers between adjacent tubules. We conclude
that 1) the principal spermatogenic lesion in TE-treated men is the
marked (90%) inhibition of type A
B spermatogonial maturation. Other
sites are also affected, particularly the release and/or survival of
elongated spermatids during transit; and 2) a steady state in germ cell
number may not be established even after 4–5 months of TE treatment.
The findings suggest that TE treatment does not adequately or
consistently withdraw hormonal support for spermatogenesis, leading to
variable between- and within-individual patterns of germ cell
suppression.
IT HAS LONG been recognized that exogenous testosterone (T) treatment, alone or in combination with progestins, reduces sperm counts in men as a result of gonadotropin suppression (1, 2, 3, 4). After the successful use of estrogen/progestins for female contraception in the 1970s, treatment with T, alone or in combination with progestins, was considered as a potential male hormonal contraceptive strategy for the effective and reversible suppression of spermatogenesis (5, 6, 7, 8, 9).
In the presence of gonadotropin suppression, T treatment is essential to prevent the loss of libido and virilization. T enanthate (TE), administered weekly at a dose of 200 mg, im, suppressed serum gonadotropin levels and induced oligo- or azoospermia in almost all subjects (5, 8). The TE alone strategy was further investigated in studies organized through the WHO (10, 11, 12, 13, 14, 15). Azoospermia was induced in 40–90% of subjects, and there was a varying degree of spermatogenic suppression seen within and between racial groups (10, 15). The basis for the different responses was unclear and not explicable on the basis of T pharmacokinetics (14) or anthropometric measures, leading to the conclusion that intrinsic differences in the spermatogenic process must underlie this heterogeneous response (12).
Although many animal studies have examined the effects of gonadotropin withdrawal on the spermatogenic process (16), this issue has not been well studied in men treated with androgens/progestins. Spermatogenesis has been reported to be suppressed to the level of spermatogonia in T-treated men based on qualitative light microscopy (3, 4, 7, 17, 18, 19). However, the pattern was not always uniform in the small number of subjects studied. Certainly no quantitative studies of germ cell populations have been performed, and as a consequence, the site(s) in the spermatogenic process inhibited by the treatment is unclear.
In the present study, we have used an unbiased and efficient
stereological technique, the optical disector, to estimate the number
of germ cells in testicular biopsy tissue after 19–24 weeks of TE
treatment. We show that germ cell numbers are greatly reduced and that
the major spermatogenic lesion is in type AB spermatogonial
development. However lesions also exists in the maturation of
spermatids and in the release and/or survival during transit of
elongated spermatids. Finally, the pattern of germ cell suppression
varies widely both between men and within individuals. Several concepts
emerge that may assist in understanding the basis of this heterogeneous
pattern of response and the morphological basis of the variable
clinical responses to T-based contraception.
Subjects and Methods
Subjects
Ten normal men presenting for vasectomy were recruited for a study in which they were to undergo testicular biopsy at the time of surgery for the evaluation of testicular morphology. The men were 38.2 ± 1.7 yr of age (range, 31–46 yr) and had fathered one to four children. The men had no history of significant illness or medication. General and testicular examination were normal, and testicular volume was determined using ultrasound. The men had at least two normal semen analyses using WHO criteria (20). Finally, the subjects all had normal serum T, LH, FSH, and PRL levels; fasting lipids; liver function tests; prostate-specific antigen; and full blood examination, except for one man (T2) with a modestly elevated serum FSH level. The study was approved by the human ethics committee of both the Monash Medical Center and the Alfred Healthcare Group, and informed consent was obtained from each subject.
Design
Five men received TE [Primotestin depot, Schering Australia;
200 mg (0.8 mL), im, weekly] for 19–24 weeks before surgery (TE
group; subjects T1–T5). The remaining five men served as controls
(subjects C1–C5) and proceeded directly to surgery. Pretreatment
clinical characteristics, semen analyses, and endocrine parameters
(Table 1
) did not differ between TE and control
groups.
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Serum T and gonadotropin levels were assessed after 11–13 weeks of TE treatment. Immediately before surgery (after 19–24 weeks of TE treatment), testicular volume was again measured using ultrasound, and a blood sample was taken for T, FSH, LH, and inhibin B levels. All blood samples were taken at random times in relation to TE injections.
Testicular biopsy and tissue processing
A single testicular biopsy was obtained from one testis after incision of the tunica. Great care was taken to avoid compression of the testis so as to minimize disruption of the testicular architecture. Each biopsy (200–500 mg) was immersion fixed in Bouin’s fluid for about 3–5 h and then observed under a dissecting microscope. Under direct vision, the biopsy was divided in two along a plane lying at right angles to the dominant tubule orientation. One piece was then further processed and embedded in hydroxyethyl-methacrylate resin (Technovit 7100, Kulzer Co., Friedrichsdorf, Germany) according to the manufacturer’s instructions. A 25-µm-thick section with an average area of about 22 mm2 was cut from each block using a Supercut microtome (Reichert Jung 2050, Nossloch, Germany), stained with periodic acid-Schiff’s reagent, and counterstained with hematoxylin. Thin 2- to 3-µm sections were prepared in a similar way for micrography.
Immunocytochemical determination of immature spermatids in semen
Semen samples were washed in phosphate-buffered saline (0.01 mol/L; pH 7.2), and smears were immunostained using monoclonal antibodies against the human intraacrosomal antigen SP-10 (SP-10-3 mAbs) (Virginia Biotech, VI) and the human leukocyte common antigen CD45 (T29/33, Dako, Carpenteria, CA) to allow the identification of immature (round) spermatids and leukocytes as previously described (21).
Tubule diameter and epithelial appearance
An approach was used similar to that previously described for the quantitative assessment of primate spermatogenesis (22). Tubule cross-sections, with an elliptical profile and either a clear lumen or a central cytoplasmic area, were sampled, as such features indicate that a complete tubule cross-section is represented. The short axis (tubule diameter) was measured. The presence of spermatocytes or spermatids in each tubule profile was recorded.
Estimation of germ and Sertoli cell number
Germ cells and Sertoli cells were identified based on their nuclear morphology (23, 24, 25, 26). Germ cells were grouped as follows: type A spermatogonia; type B spermatogonia; preleptotene, leptotene, and zygotene (Pl-Z) primary spermatocytes; pachytene (P) primary spermatocytes (including secondary spermatocytes); step 1–2 round spermatids and step 3–6 elongating spermatids, using the terminology of Sharpe (27), which equate to Sa and Sb+Sc classes as described by Clermont (23, 24, 25); and step 7–8 (Sd) late elongated spermatids.
Germ cell and Sertoli number were estimated using the optical disector technique (28, 29) as previously described for the monkey testis (22). In brief, germ and Sertoli cell nuclei or nucleoli were counted in thick methacrylate sections by superimposing a computer-generated unbiased counting frame on a video image of the section. The objective (Olympus Splan Apo x100, Olympus Corp., Lake Purchase, NY) used had a high numerical aperture (NA=1.4), which gives a very short depth of focus. Using this objective, a known depth within the section was optically sectioned, and nuclei or nucleoli were counted according to a three-dimensional counting rule (28) as they came into sharp focus. Displacement in the z-axis was monitored using a microcator (Heidenhain, Tranreut, Germany) with its sensor monitoring the stage movement; this instrument measures displacement with an accuracy of less 0.5 µm.
Germ cell data are primarily expressed on a per 100 Sertoli cells but also on a per mm3 tubule basis. Finally, data are also expressed on a per testis basis using the testicular volume; however, these calculations assume that no significant artifacts in testicular architecture were induced by the biopsy procedure.
Estimation of Sertoli cell nuclear volume
This was determined using a stereological approach: local vertical sections (30) according to an unbiased and efficient stereological principle, the rotator (31). Fifty Sertoli cell nuclei were sampled [by the optical disector (29)] and measured per testis (32).
Endocrine hormone assays
Serum LH and FSH were measured using enzyme-linked immunoassays [Imx LH, Abbott (North Chicago, IL), and AIA-Pack, TOSOH (Tokyo, Japan), respectively], both with sensitivities of 1 IU/L and with between- and within-assay coefficients of variation of 6.7% and 5.5%, and 5.9% and 4.0%, respectively. As serum FSH was suppressed below this level of detection during T treatment, serum taken at the time of surgery was also analyzed using the Axysym assay method (Abbott), with a detection limit of 0.1 IU/L.
Serum T was measured by RIA using a rabbit T antibody and iodinated histamine-T as tracer. The within- and between-assay coefficients of variation were 6% and 12%, and the working range was 0.3–58 nmol/L. High value samples were assayed at dilution.
Serum inhibin B levels were measured with a two-site enzyme-linked
immunoassay, using plates coated with an antibody to the ßB-subunit
and a second antibody against the 
-subunit, conjugated to alkaline
phosphatase (33). This assay had a sensitivity of 6 pg/mL, and all
samples were run in the same assay.
Statistics
Data are shown as the mean ± SEM (n = 5/group). Statistical comparison of data from control and TE-treated men was performed (using Sigmastat V1.0, Jandel Scientific Software, San Rafael, CA), using t test or Mann-Whitney rank sum test, when normality or equal variance test failed.
Results
Endocrinology
Serum T, FSH, LH, and inhibin B levels in the control and TE
groups are shown in Table 1
. No differences were seen in the baseline
data between groups. After TE treatment for 11–13 weeks, serum T
levels increased 2-fold to 43.5 ± 7.8 nmol/L, and serum FSH and
LH levels were all at or below the limits of assay detection (<1.0
IU/L).
At the time of surgery (19–24 weeks of TE), the mean serum T level was 63.2 ± 12 nmol/L. Serum FSH levels were borderline detectable in two TE-treated subjects (T3 and T4) using the second, more sensitive assay system.
Serum inhibin B levels were suppressed to 65% of control values (P < 0.05) by TE treatment and were clearly detectable in all subjects.
Semen analysis
Semen volumes were 4.4 ± 1.2 and 3.0 ± 0.5 mL in
control and TE groups, respectively, and did not change during TE
treatment. Baseline sperm counts were 132 ± 49 and 130 ± 50
million/mL (P = NS) in control and TE-treated groups,
respectively (Fig. 1). In response to TE
treatment, azoospermia was induced in four of the five subjects;
however, one subject (T4) had a sperm density of 21 million/mL (his
pretreatment sperm density was 317 million/mL) after 19 weeks of TE
treatment.
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Testicular morphology
Baseline testicular volumes were not significantly different in the control and TE groups (20.6 ± 4.0 and 16.7 ± 2.5 cm3, respectively). After 19–24 weeks of TE treatment, testis volume decreased by a mean of 28% (range, 8–41%), and tubule diameter decreased to 67% of the control value (138 ± 8.1 compared with 205 ± 3.0 µm; P < 0.05).
Germ cell numbers
Mean data. Germ cell data for control and TE-treated men are
considered on a per 100 Sertoli cells basis (Table 2
and Fig. 2a); however, the data are also
shown per mm3 tubule and per testis (Table 2). The number
of type A spermatogonia did not change in response to TE treatment on a
per Sertoli cell or per testis basis, whereas the apparent doubling in
type A spermatogonial and Sertoli cell number per mm3
tubule reflects the reduction in tubule diameter and, hence,
volume.
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A ratio was made between the number of step 3–6 spermatids per 100 Sertoli cells and the number of step 1–2 forms as an index of the efficiency of transition through spermiogenesis. This ratio was significantly reduced in TE-treated men compared with controls (0.38 ± 0.13 vs. 0.95 ± 0.28; P < 0.01). On the other hand, the ratio of step 7–8 spermatids to step 3–6 forms was significantly higher in TE-treated men (2.42 ± 1.94 vs. 0.64 ± 0.23; P < 0.05).
Individual data. Although in all cases a marked reduction in
type B spermatogonia and later germ cells types was apparent, there was
considerable variability in the extent of suppression of type B
spermatogonial and later germ cell number between individuals (Fig. 2, b–f). Furthermore, there was no clear relationship between 1) sperm
counts and the site and degree of spermatogenic inhibition, and 2) germ
cell numbers and the time required to achieve a sperm count below 0.1
million/mL. Specific features include the following.
Relationship between germ cell numbers and time to
azoospermia: In subjects T1 and T2 (Fig. 2, b and c), similarly
marked reductions in type B spermatogonia and all later germ cell
numbers were seen despite their times to azoospermia differing markedly
(21 vs. 8 weeks, respectively). Subject T3 (Fig. 2d)
achieved azoospermia within 6 weeks of TE treatment, yet his total
spermatocyte and spermatid populations were 2–4 times greater than
those of T1, who did not achieve azoospermia until 21 weeks.
Variability in germ cell complement within azoospermic men:
Subjects T2 and T3 (Fig. 2, c and d) were rendered azoospermic over a
similar time course (6–8 weeks); however, total spermatocytes and
spermatid numbers were 32- and 14-fold greater in T3, respectively.
Correlation between sperm and germ cell counts: Subject T4
showed suppression to 21 million/mL (
7% of his baseline) consistent
with the persistence of about 25% control levels of spermatocytes and
spermatids, yet similar precursor cells numbers (including elongated
spermatids) were seen in subject T5 who was azoospermic (by 15
weeks).
Presence of elongated spermatids in the testes of azoospermic men: In the four azoospermic men (T1–T3 and T5), elongated spermatids persisted in the testis at 1.4–20% of control levels.
Presence of spermatocyte/spermatid numbers greater than type B
spermatogonia: In subjects T1 and T3, there was a virtual absence
of type B spermatogonia, yet greater populations of later germ cell
types were seen, e.g. step 1–6 spermatids were 3.3% and
10.5% of the control value, respectively (Fig. 2, b and d).
Heterogeneity between tubules within men: In all TE-treated
men, tubule diameter and lumen were marked reduced compared to control
values (Fig. 3). In two azoospermic men
(T1 and T2), most tubule profiles contained predominantly Sertoli cells
and spermatogonia (Fig. 3B); however, there was marked heterogeneity,
in that 76% and 14%, respectively, of tubule profiles showed at least
some spermatocytes and/or spermatids (Fig. 3C). In the two remaining
azoospermic subjects (T3 and T5), more than 97% of tubule profiles
contained spermatocytes and/or spermatids (Fig. 3D). Even in the
subject (T4) with a sperm density of 21 million/mL, a few tubules
containing only Sertoli cells and spermatogonia were seen juxtaposed to
tubules showing only mild hypospermatogenesis (not shown).
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The total Sertoli cell number per testis was not affected by TE
treatment (Table 2). In response to TE treatment, the Sertoli cell
number per mm3 tubule was significantly (P
< 0.05) increased. Sertoli cell nuclear volume was significantly
(P < 0.05) reduced by 22% after TE treatment
(260 ± 29 compared to 332 ± 25 µm3 in
controls).
Discussion
T-based contraceptive treatments induce azoospermia in approximately 70% of Caucasian men, whereas the remainder have variable degrees of oligospermia and may remain fertile (10, 11). This heterogeneous response remains unexplained as both azoo- and oligospermic men show similar T pharmacokinetics and degrees of gonadotropin suppression, leading to the speculation that intrinsic differences exist in the sensitivity of the spermatogenic process to hormonal withdrawal between men (12). The degree of spermatogenic suppression in such studies was assessed using sperm counts that, although representing a useful indicator of overall spermatogenic response and fertility potential, cannot contribute to the understanding of the process by which spermatogenesis is variably affected by T treatment.
In this study we used open testicular biopsy and modern stereological approaches to estimate germ cell number so as to identify the site(s) of inhibition of the spermatogenic process in normal men undergoing T-based contraceptive treatment as previously described (10, 11). Our study is the first for two decades to describe the effects of androgen-induced gonadotropin withdrawal on the human testis. Earlier descriptive studies used nonquantitative assessments to conclude that the seminiferous epithelium regressed to the spermatogonial stage in response to a range of steroids, including medroxyprogesterone acetate alone (34) or with T (18), or combinations of danazol and T (35).
We have shown that the most consistent lesion induced by T treatment is the reduction of type B spermatogonial number to about 10% of the control value. Even though the total number of A spermatogonia remained unchanged, it is not possible to conclude whether the reduction in B forms was due to a reduction in the final division of type A spermatogonia and/or to the loss of type B forms. Inhibition of spermatogonial division may still be occurring, but may involve only a small fraction of the Apale subclass and, therefore, may not be apparent when all type A forms are pooled during quantification. Improved methods of spermatogonial identification and categorization are required to address this issue. We observed a similarly striking reduction in type B spermatogonial number in response to GnRH antagonist-induced gonadotropin withdrawal in primates (36).
Our data make it clear that other spermatogenic sites are also affected. In TE-treated men, the relatively greater decline in step 3–6 compared to step 1–2 spermatid number suggests a reduced efficiency of midspermiogenic maturation, like that seen in rodents (37), although to a lesser degree. More striking, however, was the relative excess of step 7–8 compared to step 3–6 spermatids, suggesting that elongated spermatids accumulate in the epithelium due to their impaired release. All TE-treated men had elongated spermatids present in the epithelium, yet in only one case did they appear in the ejaculate. Oligospermia induced by medroxyprogesterone acetate (34) or combinations of danazol and T (35) has been reported to be associated with an increase in round (immature) spermatids in the semen, suggesting that spermatid sloughing may be part of the mechanism of T-induced spermatogenic failure, as clearly shown in rats (37). However, we did not observe any increase in immature spermatids in this study or in a previous one involving treatment with T plus progestins (21).
There was a highly variable pattern of response seen between men in terms of both the timing of onset of azoospermia and the degree of spermatogenic inhibition. Finally, we noted striking differences in spermatogenic status between tubules within the same individual, an observation that would remain unrecognized if only assessing the response using semen analysis. Such heterogeneity also underlines the need to use a systematic uniform random sampling scheme when undertaking quantitative studies (32).
The between-individual heterogeneity requires further discussion. In
two men (T1 and T2), the germ cell patterns were consistent with a
severe lesion at the B spermatogonial stage and normal subsequent
progression up to the elongated spermatid stage, but then a failure of
these to appear in the ejaculate. A more striking example of late
spermiogenic failure was seen when comparing two other men (T4 and T5).
They had a similar and more modest reduction in type B number (25%
control), and their subsequent germ cell progression was again normal
up to the elongated spermatid stage, but there was a total failure of
their appearance in the ejaculate in T5, whereas T4 achieved a sperm
density of 21 million/mL.
Finally, the data from subject T3 suggests that a steady state of germ
cell development had not been reached despite 5 months of T treatment
(or 2 spermatogenic cycles). He had very few type B spermatogonia
(<1%), yet his spermatocyte and spermatid numbers remained 11–27%
of the control value. The only explanation for this pattern would be
that a steady state had not yet been achieved and that a late-onset
severe depletion in type B would shortly flow on to reduce these later
cell types. This proposition is supported by the clinical observation
that a substantial proportion of men who are oligospermic after 4–6
months of T treatment alone do, in fact, become azoospermic over the
next 6 months (11).
These different patterns of response did not relate to the time to azoospermia or the degree of suppression of inhibin B (as a marker of Sertoli cell function). Inhibin B levels were suppressed by about one third, similar to those reported by Anawalt et al. (33), but were clearly detectable in all subjects, underlining the gonadotropin independence of a substantial proportion of inhibin B secretion in men. Serum FSH levels were at or below the limit of assay sensitivity in the four azoospermic men, but were just detectable in subject T4, with 21 million/mL sperm count. Whether a low persistent level of FSH in any way was contributing to his continued spermatogenesis is a moot point.
The optical disector stereological approach is an unbiased method
unaffected by the shape of cells or nuclei/nucleoli. In our animal
studies we expressed our data on a per testis basis, as the reference
volume of the testis was determined and the biopsies were taken from
multiple sites after fixation. Obviously this was not possible in the
current study, and we, therefore, expressed germ cell number per 100
Sertoli cells based on the assumption that Sertoli cell number was not
affected by treatment. The validity of this assumption has been tested
in the GnRH-deficient primate (36) and in rats (38). Nonetheless, we
also cautiously expressed the data per testis and in doing so have
assumed that the biopsy was not subject to significant distortion or
shrinkage (particularly the tubule/interstitial relationship) during
collection and processing. Our estimate of total Sertoli cell number
per testis (760 million) is about half that reported by Cortes
et al. (1350 million) (39), but is in reasonable agreement
with that reported by Johnson et al. (500 million) (40).
The approach to sampling the human testis was necessarily limited to the use of a single open biopsy. The human testis is comprised of 200–300 lobules, each containing 1–3 highly coiled tubules approximately 70–80 cm in length. We estimate that at most we would have sectioned through 6 individual tubules. However, it is impossible to determine the origin of any particular tubule profile or its relationship to adjacent tubules. Accordingly, one cannot conclude whether the variable degree of spermatogenic inhibition seen in adjacent profiles is due to the persistence of spermatogenesis in one entire tubule or arises from patchy maintenance within several tubules.
How can a variable spermatogenic state exist between tubules or across tubules within one man in whom the endocrine signals ought be identical? Even the proposition that the paracrine environment might vary so substantially between adjacent tubules seems difficult to sustain. Although not being able to directly address the mechanism of this heterogeneity, one can speculate that such a pattern might be produced when the withdrawal of the hormonal support for spermatogenesis achieved using the TE alone protocol is a borderline effective approach. In an analogous fashion to a chemotherapeutic action on tumor cells, the use of a borderline effective treatment will result in a range of responses within a presumably monoclonally derived cell population, i.e. cells may be killed, cease dividing (temporarily or permanently), or be unresponsive. In the case of spermatogenesis, it should not be surprising that some tubule sections or Sertoli/germ cell units can continue to function while the process ceases in adjacent areas. With the passage of time, some regions adapt to provide long term sperm production (persistent oligospermia), whereas spermatogenesis in other regions ultimately collapses.
To extend the analogy with chemotherapy further, the more effective the treatment, the more rapid and consistent the outcome, e.g. the use of combination chemotherapies. Indeed, in contraception, the use of T in combination with other agents, such as GnRH antagonists (41) and progestins (42, 43, 44), has been reported to achieve a more consistent and rapid induction of azoospermia. In the rat, the inclusion of the androgen receptor antagonist, flutamide, with a GnRH antagonist results in the accelerated depletion of elongated spermatids (45). The common feature of these approaches is the more rapid and profound suppression of gonadotropin levels and/or the inhibition of residual androgen action within the testis.
The nonhomogeneity of the human testis is also apparent from the recent demonstration that elongated spermatids can be isolated from open testicular biopsy in men with spermatogenic failure in whom thorough conventional histology reveals the pattern of the Sertoli cell only syndrome or germ cell arrest at the spermatocyte stage (46). Clearly, whatever genetic/environmental damage exists does not apply evenly throughout the testis. It has been suggested that the isolation of sperm from men with severe hypospermatogenesis in whom some elongated spermatids are seen indicates that their azoospermia results from a coexistent obstructive lesion. Our data in the T-treated man clearly shows that azoospermia can result from the failure of elongated spermatids to be released and/or to survive transit into the ejaculate. The mechanism of this failure requires further study.
In conclusion, we have shown that the principal spermatogenic lesion in
TE-treated men is the inhibition of type AB spermatogonial
maturation; however, other sites are affected, particularly the release
and/or survival of elongated spermatids. The wide variability in the
timing and degree of germ cell depletion suggests that TE treatment
does not lead to a steady state even after 5 months of TE treatment. We
speculate that TE treatment fails to provide effective and consistent
withdrawal of hormonal support for spermatogenesis, which, in turn,
leads to the variable between- and within-individual patterns of germ
cell suppression.
Acknowledgments
June Lee is thanked for her assistance in conducting many aspects of the study, Ms. J. Spaliviero and Dr. D. Handelsman for the performance of the sensitive FSH assay, Dr. J. Doery and the Biochemistry Department of the Monash Medical Center, and Mr. N. Cahir and Dr. David Robertson for the performance of the inhibin B assay and for helpful comments on the manuscript. The kind provision of Primotestin depot by Schering Australia is gratefully acknowledged.
Footnotes
1 This work was supported by the Contraception 21 Grant of the
Rockefeller Foundation, Australian National Health and Medical Research
Council Grants 943208 and 973218, Australian Research Council Grant
80900657, and Schering Australia. 
Received November 13, 1997.
Revised December 31, 1997.
Accepted January 8, 1998.
References
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Y. Lue, C. Wang, Y.-X. Liu, A. P. S. Hikim, X.-S. Zhang, C.-M. Ng, Z.-Y. Hu, Y.-C. Li, A. Leung, and R. S. Swerdloff Transient Testicular Warming Enhances the Suppressive Effect of Testosterone on Spermatogenesis in Adult Cynomolgus Monkeys (Macaca fascicularis) J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 539 - 545. [Abstract] [Full Text] [PDF]
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C. Garrett, D.Y. Liu, R.I. McLachlan, and H.W.G. Baker Time course of changes in sperm morphometry and semen variables during testosterone-induced suppression of human spermatogenesis Hum. Reprod., November 1, 2005; 20(11): 3091 - 3100. [Abstract] [Full Text] [PDF]
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K. L. Matthiesson, P. G. Stanton, L. O'Donnell, S. J. Meachem, J. K. Amory, R. Berger, W. J. Bremner, and R. I. McLachlan Effects of Testosterone and Levonorgestrel Combined with a 5{alpha}-Reductase Inhibitor or Gonadotropin-Releasing Hormone Antagonist on Spermatogenesis and Intratesticular Steroid Levels in Normal Men J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5647 - 5655. [Abstract] [Full Text] [PDF]
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 Y. Wang, H. Newton, J. A. Spaliviero, C. M. Allan, B. Marshan, D. J. Handelsman, and P. J. Illingworth Gonadotropin Control of Inhibin Secretion and the Relationship to Follicle Type and Number in the hpg Mouse Biol Reprod, October 1, 2005; 73(4): 610 - 618. [Abstract] [Full Text] [PDF]
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S. J. Meachem, P. G. Stanton, and S. Schlatt Follicle-Stimulating Hormone Regulates Both Sertoli Cell and Spermatogonial Populations in the Adult Photoinhibited Djungarian Hamster Testis Biol Reprod, May 1, 2005; 72(5): 1187 - 1193. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, A. Bae, R. S. Swerdloff, A. Iranmanesh, and C. Wang Experimentally Induced Androgen Depletion Accentuates Ethnicity-Related Contrasts in Luteinizing Hormone Secretion in Asian and Caucasian Men J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1632 - 1638. [Abstract] [Full Text] [PDF]
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B.M. Brady, M. Walton, N. Hollow, A.T. Kicman, D.T. Baird, and R.A. Anderson Depot testosterone with etonogestrel implants result in induction of azoospermia in all men for long-term contraception Hum. Reprod., November 1, 2004; 19(11): 2658 - 2667. [Abstract] [Full Text] [PDF]
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J. Wistuba, A. Schrod, B. Greve, J. K. Hodges, H. Aslam, G. F. Weinbauer, and C. M. Luetjens Organization of Seminiferous Epithelium in Primates: Relationship to Spermatogenic Efficiency, Phylogeny, and Mating System Biol Reprod, August 1, 2003; 69(2): 582 - 591. [Abstract] [Full Text] [PDF]
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K. L. Matthiesson, D. M. Robertson, H. G. Burger, and R. I. McLachlan Response of serum inhibin B and pro-{alpha}C levels to gonadotrophic stimulation in normal men before and after steroidal contraceptive treatment Hum. Reprod., April 1, 2003; 18(4): 734 - 743. [Abstract] [Full Text] [PDF]
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W.M. Hair, F. C.W. Wu, and G. A. Lincoln An investigation of the effectiveness of testosterone implants in combination with the prolactin inhibitor quinagolide in the suppression of spermatogenesis in men Hum. Reprod., April 1, 2003; 18(4): 749 - 755. [Abstract] [Full Text] [PDF]
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 R. A. Anderson and D. T. Baird Male Contraception Endocr. Rev., December 1, 2002; 23(6): 735 - 762. [Abstract] [Full Text] [PDF]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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R. I. McLachlan, L. O'Donnell, P. G. Stanton, G. Balourdos, M. Frydenberg, D. M. de Kretser, and D. M. Robertson Effects of Testosterone Plus Medroxyprogesterone Acetate on Semen Quality, Reproductive Hormones, and Germ Cell Populations in Normal Young Men J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 546 - 556. [Abstract] [Full Text] [PDF]
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 R.I. McLachlan, L. O'Donnell, S.J. Meachem, P.G. Stanton, D.M. de Kretser, K. Pratis, and D.M. Robertson Identification of Specific Sites of Hormonal Regulation in Spermatogenesis in Rats, Monkeys, and Man Recent Prog. Horm. Res., January 1, 2002; 57(1): 149 - 179. [Abstract] [Full Text] [PDF]
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T. M. Plant and G. R. Marshall The Functional Significance of FSH in Spermatogenesis and the Control of Its Secretion in Male Primates Endocr. Rev., December 1, 2001; 22(6): 764 - 786. [Abstract] [Full Text] [PDF]
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 N. G. Wreford, T. Rajendra Kumar, M. M. Matzuk, and D. M. de Kretser Analysis of the Testicular Phenotype of the Follicle-Stimulating Hormone {beta}-Subunit Knockout and the Activin Type II Receptor Knockout Mice by Stereological Analysis Endocrinology, July 1, 2001; 142(7): 2916 - 2920. [Abstract] [Full Text] [PDF]
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L. O’Donnell, A. Narula, G. Balourdos, Y.-Q. Gu, N. G. Wreford, D. M. Robertson, W. J. Bremner, and R. I. McLachlan Impairment of Spermatogonial Development and Spermiation after Testosterone-Induced Gonadotropin Suppression in Adult Monkeys (Macaca fascicularis) J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1814 - 1822. [Abstract] [Full Text]
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