help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0222
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/7/2526    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Walton, M. J.
Right arrow Articles by Anderson, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Walton, M. J.
Right arrow Articles by Anderson, R. A.
Related Collections
Right arrow Male Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 7 2526-2533
Copyright © 2006 by The Endocrine Society

Direct Effect of Progestogen on Gene Expression in the Testis during Gonadotropin Withdrawal and Early Suppression of Spermatogenesis

Melanie J. Walton, Rosemary A. L. Bayne, Ian Wallace, David T. Baird and Richard A. Anderson

Division of Reproductive and Developmental Sciences (M.J.W., D.T.B., R.A.A.), Medical Research Council Human Reproductive Sciences Unit (R.A.L.B.), Centre for Reproductive Biology, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom; and Family Planning and Well Woman Services (I.W.), Lothian Health, Edinburgh EH4 1NL, United Kingdom

Address all correspondence and requests for reprints to: Professor R. A. Anderson, Centre for Reproductive Biology, The Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail: richard.anderson{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Testicular production of steroids and gametes is under gonadotropin support, but there is little information as to the molecular mechanisms by which these are regulated in the human. The testicular response to gonadotropin withdrawal is important for the development of effective contraceptive methods.

Objective: Our objective was investigation of expression of genes in the normal human testis reflecting steroidogenesis, Sertoli cell function, and spermatogenesis after short-term gonadotropin withdrawal and the effects of activating testicular progesterone receptors.

Design and Setting: We conducted a randomized controlled trial at a research institute.

Patients: Thirty healthy men participated.

Interventions: Subjects were randomized to no treatment or gonadotropin suppression by GnRH antagonist (cetrorelix) with testosterone (CT group) or with additional administration of the gestogen desogestrel (CTD group) for 4 wk before testicular biopsy. Gene expression was quantified by RT-PCR.

Results: Both treatment groups showed similar suppression of gonadotropins and sperm production and markedly reduced expression of steroidogenic enzymes. Addition of progestogen in the CTD group resulted in reduced expression of 5{alpha}-reductase type 1 compared with both controls and the CT group. Inhibin-{alpha} and the spermatocyte marker acrosin-binding protein were significantly lower in the CTD but not CT groups, compared with controls, but did not differ between treated groups. Men who showed greater falls in sperm production also showed reduced expression of these three genes but not of the spermatid marker protamine 1.

Conclusions: These data provide evidence for direct progestogenic effects on the testis and highlight steroid 5{alpha}-reduction and disruption of spermiation as important components of the testicular response to gonadotropin withdrawal.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
TESTICULAR FUNCTION IS dependent on trophic support from the gonadotropins LH and FSH. The effects of testosterone and FSH are mediated through the Sertoli and peritubular cells because these cells, but not germ cells, express receptors for these hormones (1, 2, 3). Selective withdrawal and replacement of LH (and thus intratesticular testosterone) and FSH has shown that both are required for quantitatively normal spermatogenesis (4, 5), but the pathways through which testosterone and FSH support spermatogenesis remain uncertain.

Understanding of gonadotropin-dependent testicular pathways is also important for the development of novel male contraception. Gonadotropin suppression by the administration of testosterone alone or with a progestogen or GnRH antagonist results in suppression to azoospermia in most men (6, 7). There is, however, variation in the degree of suppression between individuals (8, 9, 10). One potential mechanism for this is activity of the enzyme 5{alpha}-reductase, which converts testosterone to the more potent androgen dihydrotestosterone (11, 12).

The addition of a progestogen to testosterone-based regimens increases spermatogenic suppression (13, 14). This is greater than can be accounted for by the degree of gonadotropin suppression, providing indirect evidence that progestogens act directly on the testis (15). Expression of both nuclear and membrane progesterone receptors has been demonstrated by spermatozoa and Sertoli and some Leydig cells in the human testis (16), and progestogens have direct inhibitory effects on Leydig cell function in a murine cell line (17).

We have investigated changes in gene expression in the human testis in response to gonadotropin withdrawal. The study was also designed to investigate potential testicular effects of addition of a progestogen and correlation with spermatogenic suppression. Chosen genes reflected the various testicular compartments, i.e. steroidogenesis, Sertoli cell activity, and germ cells at different stages of maturation.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patient recruitment

Thirty healthy men requesting vasectomy were recruited. Inclusion criteria included age (18–50 yr), no past significant medical problems, normal biochemical and hematological parameters, normal andrological examination, sperm concentration greater than 20 x 106/ml, and normal plasma LH, FSH, and testosterone concentrations. All subjects provided written informed consent. The study had ethical approval from the Lothian Regional Ethics Committee and was performed according to Good Clinical Practice guidelines.

Study design and drug treatment

The study was a randomized controlled trial of the effects of cetrorelix and testosterone with or without desogestrel. After satisfactory completion of screening examination and investigations, 30 subjects were randomly allocated to one of three treatment groups by third-party randomization using sealed envelopes in blocks of 10. Ten subjects were allocated to the control group and had a testis biopsy at the time of vasectomy without study drug administration. Ten subjects were treated with cetrorelix and testosterone (CT group), and the other 10 were given the progestogen and desogestrel in addition to the other agents (CTD group). Cetrorelix (Cetrotide; Serono Europe Ltd., London, UK), 3 mg sc, was administered twice each week. Testosterone was administered as testosterone enanthate (Cambridge Laboratories, Wallsend, UK), 200 mg im, on the first day of administration of cetrorelix and repeated 14 d later. The 10 men in the CTD group also took desogestrel, 300 µg orally (Cerazette, 4 x 75 µg; Organon NV, Oss, The Netherlands) each day for the 28-d duration of the treatment period.

Biopsy

Testis biopsy was carried out under local anesthetic at the time of vasectomy. The lower pole of one testis was biopsied using two to four passes of a 14-gauge needle (Tru-Cut; Allegiance Healthcare Corp., McGaw Park , IL) as reported (18). Tissue samples were immediately frozen and stored at –80 C. Because of the nature of the biopsy, specimens were not suitable for histological examination.

Isolation of RNA and synthesis of cDNA

Total RNA was extracted using the QIAGEN RNeasy Mini kit. High RNA quality (RNA integrity no. > 6.5) was confirmed using RNA 6000 Nanochips in the Agilent 2100 Bioanalyzer (Agilent Technologies, South Queensferry, UK). One sample from the CTD group and one from the control group were found to be of inadequate quality and not analyzed further. First-strand cDNA (with or without reverse transcriptase) was synthesized from 3 µg total RNA as described previously (19).

Real-time quantitative PCR

Quantitative real-time RT-PCR was performed using the Lightcycler (Roche Diagnostics, East Sussex, UK) as described previously (19). Reverse-transcribed RNA samples were diluted 1/25 in nuclease-free water (Promega Ltd., Southampton, UK). One microliter of diluted first-strand cDNA was added to a final volume of 10 µl containing 50 µg/ml BSA and 0.5 µM each of forward and reverse primer in 1x Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, Paisley, UK) in duplicate. Primers (Table 1Go) were either previously published or designed using online Primer3 software. mRNA concentration was calculated relative to the ribosomal protein RPL32. RPL32 proved to be the most consistent reference gene for these samples although GAPD and B2M (20) yielded similar results.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Sequences of primers used for quantitative PCR

 
Semen analysis

Sperm concentration in the ejaculate was measured on one occasion before treatment and again on the day of vasectomy, in both cases after 3–7 d abstinence (21). Azoospermia was confirmed by thorough examination of the pellet after centrifugation of the whole semen sample.

Hormone assay

Blood samples were obtained before treatment and on the day of vasectomy. An additional sample was taken from the men in the two treatment groups after 14-d drug administration. Testosterone was measured by RIA (22) and FSH and LH by time-resolved immunofluorometric in-house assay (23). Assay sensitivity was 0.3 nmol/liter for testosterone, 0.03 IU/liter for FSH, and 0.15 IU/liter for LH. Intraassay coefficients of variation were less than 10% in each case, and all samples were analyzed in single assays.

Statistical analysis

Results are presented as mean ± SEM. Hormone and sperm data were compared using paired or unpaired t tests as appropriate after log and cubed-root transformation, respectively. Treatment effects on gene expression data were initially compared by ANOVA (three groups) or unpaired t test (two groups). Where ANOVA suggested significant treatment-group effects, this was further investigated by unpaired t tests. For all comparisons, a P value of <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Subjects, withdrawals, and adverse events

Thirty men (mean age, 38 yr; range, 30–47 yr) were recruited to the study. There were no significant differences between treatment groups in age or pretreatment sperm and reproductive hormone concentrations (Table 2Go). One subject randomized to the control group withdrew from the study for personal reasons; thus, 29 men underwent testis biopsy (Fig. 1Go). There were no significant adverse events. Two subjects complained of itching/redness at the site of cetrorelix injections, and one subject in the CTD group reported mood swings and hot flushes during the treatment period.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Pretreatment characteristics of subjects

 

Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. Treatment pathway.

 
Hormones

Gonadotropin concentrations were markedly suppressed in all men in both treatment regimens (P < 0.001) (Fig. 2Go, A and B). Suppression after 14 d treatment was similar (data not shown). There were no differences in the concentrations of either LH or FSH between the CT and CTD groups at either time point. FSH was suppressed to the limit of detection or close to it in all men, and LH was suppressed to 0.52 ± 0.09 and 0.51 ± 0.07 IU/liter in the CT and CTD groups, respectively, at the time of biopsy.


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. Serum concentrations of LH (A), FSH (B), testosterone (C), and sperm concentration (D) in untreated controls (white bars) and men receiving cetrorelix and testosterone (CT group, black bars) or cetrorelix, testosterone, and desogestrel (CTD group, gray bars). Results are means ± SEM; n = 8–10 per group.

 
Mean testosterone concentrations remained in the normal range in the two treatment groups but were slightly reduced at 28 d (Fig. 2CGo) because this was the trough after the second injection. There were no significant differences in testosterone concentrations between the three groups either before treatment or at the time of testis biopsy.

Sperm concentrations

Pretreatment sperm concentrations were normal in all subjects. Marked suppression of sperm concentration was seen in both treatment groups (Fig. 2DGo). Mean concentrations fell to 17.8 ± 9.2 x 106/ml (P = 0.005; median, 0.6 x 106/ml) in the CT group and to 8.1 ± 4.1 x 106/ml (P < 0.0001; median, 2.6 x 106/ml) in the CTD group compared with pretreatment concentrations of 47.5 ± 8.2 and 59.9 ± 13.8 x 106/ml, respectively. However, there was some interindividual variability with three subjects in the CT group and two in the CTD group maintaining sperm concentrations in the normal range (>20 x 106/ml) at d 28, whereas sperm concentrations were less than 5 x 106/ml in all others. One man in each group had become azoospermic. This allowed classification of suppressors vs. nonsuppressors, the former having sperm concentrations less than 5 x 106/ml after 4-wk treatment, the latter having normal sperm concentrations at that time.

Testicular biopsy results

Testicular tissue specimens were analyzed by treatment vs. control, by treatment group, and by the degree of spermatogenic suppression.

Steroidogenic genes

The two genes expressing the steroidogenic enzymes steroid 17-{alpha}-hydroxylase/17,20 lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2) showed markedly reduced expression in both treatment groups (both P < 0.0001), to approximately 3 and 25% of the control group, respectively (Fig. 3AGo). This effect was similar in the two treatment groups. Expression of 5{alpha}-reductase type 1 (SRD5A1) was also reduced in both treatment groups overall compared with controls (P = 0.007) (Fig. 3Go), but analysis by group demonstrated that the reduction was greater in the CTD group (57% of controls, P = 0.0065), whereas expression in the CT group was not significantly lower than controls (77%, P = 0.1). There was also a significant difference in SRD5A1 gene expression between the CTD and CT groups, being 26% lower in the CTD group (P = 0.02).


Figure 3
View larger version (30K):
[in this window]
[in a new window]
  		FIG. 3. Expression of steroidogenic genes CYP17A1, HSD3B2, and SRD5A1 (A); Sertoli cell genes INHA, AR, AMH, FSHR, hPEPP1, and hPEPP2 (B); and germ cell genes MAGEA4, ACRBP, and PRM1 (C) in controls (white bars) and men in CT (black bars) and CTD groups (gray bars). Results are means ± SEM; n = 8–10 per group. Gene expression is relative to RPL32. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. controls; {ddagger}, P < 0.05, CTD vs. CT group.

 
Sertoli cell genes

Expression of inhibin {alpha}-subunit (INHA) was reduced in both treatment groups together (P = 0.007) (Fig. 3BGo), but this was more marked in the CTD group (42% of control, P = 0.037), with the difference between the control and CT group not quite reaching statistical significance (49% of controls, P = 0.056). There was no significant difference between the CT and CTD treatment groups. There were no differences in expression of the genes encoding the androgen receptor (AR), FSH receptor (FSHR), or anti-Müllerian hormone (AMH) between the treatment and control groups. Expression of hPEPP1 was also unchanged, whereas there was a reduction in hPEPP2 expression in the two treatment groups combined (P = 0.05), which did not reach statistical significance in either group separately [74% (P = 0.2) and 65% (P = 0.08) of controls in the CT and CTD groups, respectively].

Germ cell genes

Three genes specific to germ cells were investigated (Fig. 3CGo). Expression of MAGEA4 was reduced in the CTD group to 53% of controls, but this did not reach statistical significance (P = 0.06). MAGEA4 expression was not reduced in the CT group (75% of control, P = 0.04). Expression of ACRBP was reduced (P = 0.02). This was confined to the CTD group (61% of controls, P = 0.008) with no difference between the CT and control groups (79% of controls, P = 0.1). Expression in the CT and CTD groups was not significantly different. Expression of PRM1 was similar to control in both treatment groups.

Gene expression analyzed by sperm suppression

Subjects were classified according to the degree of spermatogenic suppression independent of treatment group, with most men (n = 14) showing marked spermatogenic suppression (sperm concentration <5 x 106/ml) even within the short treatment interval, whereas others (n = 5) maintained sperm concentrations within the normal range. There were no differences in sperm, LH, or FSH concentrations before treatment between the two groups or in LH and FSH concentrations after treatment (Table 3Go). Both groups showed markedly reduced expression of CYP17A1 and HSD3B2 compared with controls (both P < 0.0001) with no difference between suppressors and nonsuppressors. However, expression of SRD5A1 did differ between groups, with a significant difference between controls and suppressors (62% of controls, P = 0.004) but not between controls and nonsuppressors (81% of controls, P = 0.3). There was also evidence of a difference within the CT group by spermatogenic suppression (suppressors were 0.20 ± 0.01 vs. 0.30 ± 0.01 in nonsuppressors) but not in the CTD group (0.17 ± 0.03 vs. 0.16 ± 0.03, respectively), although this analysis is limited by the small group size.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Gene expression analyzed by sperm suppression

 
Among the Sertoli cell genes, the reduction in INHA expression was significant only in the suppressors (42% of controls, P = 0.01; 55% of controls in nonsuppressors, P = 0.2). hEPP2 expression was slightly reduced with borderline statistical significance in the suppressors (73% of controls, P = 0.05) but not in the nonsuppressors (79% of controls, P = 0.4). Expression of other Sertoli cell genes did not differ between controls and either group.

Markers of spermatogenesis also showed differential changes. MAGEA4 expression was only reduced in suppressors (55% of controls, P = 0.02; 93% in nonsuppressors, P = 0.8). ACRBP expression was reduced to 65% of controls in suppressors (P = 0.003) but similar to controls in the nonsuppressors (89% of control, P = 0.5). PRM1 expression did not differ from controls in either group.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
We report the effects on testicular gene expression of short-term hypogonadotropism in normal men. An additional group was administered desogestrel to investigate the effects of testicular progesterone receptor activation in addition to gonadotropin withdrawal. Importantly, the degree of gonadotropin suppression was similar in the two treatment groups at both 14 and 28 d, allowing the identification of testicular effects of desogestrel, although it is possible that there was a slightly greater suppression of gonadotropin secretion in the CTD group that we were unable to detect. Spermatogenesis was rapidly suppressed in both treatment groups and to a similar degree in the two treatment groups. Although the most marked effects were on steroidogenic enzyme expression in both treatment groups, there was evidence of a selective reduction in the testosterone-metabolizing enzyme SRD5A1 and of the spermatocyte-specific gene acrosin-binding protein (ACRBP) (24) but not of genes expressed at earlier stages of spermatogenesis in those men who were given desogestrel. The group not receiving desogestrel showed little or no differences in expression of these genes compared with the control group, despite profound suppression of gonadotropins and of spermatogenesis.

Detailed analysis of the relationships between gonadotropins and spermatogenic suppression during longer-duration suppression has previously provided indirect evidence that progestogens, including desogestrel as used here, have additional gonadotropin-independent effects (15). These data substantiate this by now providing for the first time in men direct evidence that progestogens have specific intratesticular effects independent of gonadotropin suppression, which may contribute to the enhanced suppression of spermatogenesis demonstrated in trials of hormonal male contraceptive regimens (7, 8, 23). They also illustrate the value of this approach to the study of spermatogenic suppression in response to potential contraceptive regimens and other manipulations of testicular function.

There are limited data on the use of GnRH antagonists to suppress spermatogenesis, but these results, albeit of only short treatment duration, provide support to suggestions that they are highly effective at inducing gonadotropic and spermatogenic suppression (25, 26). Although most men showed profound suppression of spermatogenesis after only 4 wk of treatment, some 25% still had sperm concentrations in the normal range. It is likely that these men would have subsequently also shown profound suppression, but our short-term study allowed a clear distinction between those showing a rapid response (here termed suppressors) and those maintaining spermatogenesis at that stage of treatment (nonsuppressors). These nonsuppressors should not be regarded as necessarily having the same characteristics as those who continue to show low rates of spermatogenesis despite more prolonged gonadotropin withdrawal, but they provide a useful group for the investigation of variation in the initial stages of spermatogenic suppression.

The most striking changes in gene expression during gonadotropin withdrawal were in the steroidogenic enzymes CYP17A1 and HSD3B2. There was no evidence for an additional effect of desogestrel and no relationship to degree of suppression of spermatogenesis. In adult animals, primary control of both enzymes is via the LH receptor (27). These data suggest that a similar mechanism of regulation occurs in humans. Progestogens have been reported to have direct effects on LH receptor expression and to inhibit steroidogenesis in murine Leydig cells (17), but the present results do not provide evidence that such regulation occurs in humans with desogestrel at the dose used here. It is, however, possible that the very pronounced suppression of CYP17A has prevented detection of an additional progestogenic effect.

The enzyme 5{alpha}-reductase converts testosterone to the more potent androgen dihydrotestosterone. This amplification of androgen action has been suggested to be of importance in the testis in states of testosterone depletion, e.g. after gonadotropin suppression, and to be a mechanism whereby some men may maintain low rates of spermatogenesis during contraceptive studies (11). This has been supported by experimental data in rodents although not by clinical studies involving inhibition of 5{alpha}-reductase (12, 28, 29, 30). In those clinical studies, however, the type 2 isoenzyme was preferentially inhibited. 5{alpha}-Reduced steroids within the testis are relatively resistant to gonadotropin withdrawal (31, 32). Progesterone and synthetic progestogens have been reported to inhibit 5{alpha}-reductase activity in skin (33, 34, 35), which predominantly contains the type 1 isoenzyme. The present data demonstrate that SRD5A1 gene expression was reduced by administration of desogestrel but not by gonadotropin suppression alone. SRD5A1 expression was also reduced in relation to the degree of suppression of spermatogenesis. This was apparent in the CT group, but no difference by spermatogenic suppression was observed in the CTD group. This may reflect the already-present inhibitory effect of desogestrel, but analysis is limited by the small group sizes. These data therefore add to the evidence implicating 5{alpha}-reduction as a key pathway in the testicular response to hormonal contraceptive regimens, particularly those based on coadministration of a progestogen.

Despite the degree of gonadotropin withdrawal and fall in sperm production, there were few changes in expression of the Sertoli cell genes investigated. Expression of INHA (encoding inhibin {alpha}), also expressed by Leydig cells (36, 37), was modestly reduced and was statistically significant only in the CTD group. Additionally, analysis by degree of sperm suppression showed lower INHA expression only in the suppressors group. This may indicate both a direct effect of progestogen on Sertoli cell function in keeping with the expression of progesterone receptors by human Sertoli cells (16) and a relationship with spermatogenesis. Inhibin B is the biologically active form in men consisting of a dimer of the {alpha}- and ßB-subunits, its concentration in blood quantitatively reflecting spermatogenesis (38). Gonadotropin withdrawal results in a fall in inhibin B but only to approximately 50% of normal, even with prolonged treatment (39). This appears to match the change in gene expression observed here, consistent with the remaining translation and transcription being gonadotropin independent in normal men.

PEPP1 and PEPP2 are the human homologs of the murine Pem gene, which is highly androgen dependent (40, 41). These data confirm that PEPP1 and PEPP2 are expressed in the human testis and demonstrate that expression of PEPP2 was reduced in treated men compared with controls. Expression of PEPP2 was also reduced in the spermatogenic suppressor group but not in nonsuppressors. These data are consistent with PEPP2 expression being androgen dependent but, as in the rat, less androgen dependent than in the mouse (41), emphasizing the importance of species specificity in the regulation of testicular function. Expression of other Sertoli cell genes, FSHR, AR, and AMH, was similar in controls and treated groups. This may indicate that expression of these genes is not closely gonadotropin dependent or linked to normal spermatogenesis but may reflect the relatively short duration of gonadotropin withdrawal.

MAGEA4 is expressed by spermatogonia and primary spermatocytes but not by spermatids or Sertoli cells (42) and is therefore a marker for early spermatogenesis. We also investigated expression of ACRBP as an additional spermatocyte-specific marker (24). MAGEA4 expression was similar in the control to the two treatment groups but was reduced in the suppressor group. ACRBP showed greater changes, being reduced in the CTD but not CT groups compared with controls, indicating a direct effect of the progestogen. As with MAGEA4, there was markedly lower ACRBP expression in men in the suppressor group but not in nonsuppressors. The greater changes in ACRBP than MAGEA4 may reflect greater changes in spermatocytes than spermatogonia. This is consistent with data from stereological analysis of testis biopsies (31) following similar treatment regimens to that used here. Those data indicated that after only 2 wk of testosterone/progestogen treatment, there were fewer type B spermatogonia and early spermatocytes present in the seminiferous epithelium. Changes in gene expression in spermatogonia and spermatocytes therefore occur rapidly after gonadotropin withdrawal and are associated with more rapid suppression of spermatogenesis.

In contrast to these changes in markers of early and mid stages of spermatogenesis, there were no differences in expression of PRM1 either by treatment group or by rate of spermatogenic suppression. PRM1 is expressed in postmeiotic haploid spermatids (43, 44). The maintenance of PRM1 expression here is striking considering the dramatic reduction in sperm output in most men in the treatment groups. These data therefore strongly support the observation that one of the major early defects in the human spermatogenic epithelium after gonadotropin withdrawal is an inhibition of spermiation (31, 32) resulting in retention of the cell types expressing PRM1 while at the same time drastically reducing sperm output. It also is in keeping with the rapid return of sperm to the ejaculate in some men after withdrawal or brief omission of hormonal contraception (10). This is similar to findings in the macaque monkey and rodents, in which gonadotropin withdrawal also results in retention of elongate spermatids (45, 46). With longer gonadotropin withdrawal, all spermatogenic cell types are reduced (31) and would be expected to result in a decrease in PRM1 expression.

In conclusion, these data demonstrate the value of a novel approach to the investigation of testicular function in men. We have presented direct evidence for progestogenic effects on all three major compartments of the testis, i.e. steroidogenesis (5{alpha}-reductase), Sertoli cell function (inhibin {alpha}), and spermatogenesis (ACRBP), independent of gonadotropin inhibition. Furthermore, relationships between expression of these and other genes (PEPP2 and MAGEA4) and the rate of initial suppression of sperm output were identified. Notably, these data provide additional support for the importance of 5{alpha}-reduction and disruption of spermiation as important components of the testicular response to gonadotropin withdrawal, of direct relevance to understanding of testicular function and the development of novel methods of male contraception.


   Acknowledgments
 
We are grateful to Ann Kerr for assistance with patient recruitment, Neil Hollow for hormone analysis, Anna Glasier for encouragement and provision of facilities to use this model, and Richard Sharpe and Philippa Saunders for helpful discussions. We also acknowledge support from Serono UK toward the provision of cetrorelix.


   Footnotes
 
This work was supported by a grant from the Medical Research Council and Department for International Development (G9523250).

First Published Online April 18, 2006

Abbreviations: CT, Cetrorelix and testosterone; CTD, CT plus desogestrel.

Received January 31, 2006.

Accepted April 10, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Collins LL, Lee HJ, Chen YT, Chang M, Hsu HY, Yeh S, Chang C 2003 The androgen receptor in spermatogenesis. Cytogenet Genome Res 103:299–301[CrossRef][Medline]
  2. Bremner WJ, Millar MR, Sharpe RM, Saunders PT 1994 Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135:1227–1234[Abstract]
  3. Themmen APN, Huhtaniemi IT 2000 Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev 21:551–583[Abstract/Free Full Text]
  4. Matsumoto AM, Karpas AE, Paulsen CA, Bremner WJ 1983 Reinitiation of sperm production in gonadotropin-suppressed normal men by administration of follicle-stimulating hormone. J Clin Invest 72:1005–1015[Medline]
  5. Matsumoto AM, Paulsen CA, Bremner WJ 1984 Stimulation of sperm production by human luteinizing hormone in gonadotropin-suppressed normal men. J Clin Endocrinol Metab 59:882–887[Abstract]
  6. Anderson RA, Baird DT 2002 Male contraception. Endocr Rev 23:735–762[Abstract/Free Full Text]
  7. Nieschlag E, Zitzmann M, Kamischke A 2003 Use of progestins in male contraception. Steroids 68:965–972[CrossRef][Medline]
  8. Turner L, Conway AJ, Jimenez M, Liu PY, Forbes E, McLachlan RI, Handelsman DJ 2003 Contraceptive efficacy of a depot progestin and androgen combination in men. J Clin Endocrinol Metab 88:4659–4667[Abstract/Free Full Text]
  9. Kamischke A, Heuermann T, Kruger K, von Eckardstein S, Schellschmidt I, Rubig A, Nieschlag E 2002 An effective hormonal male contraceptive using testosterone undecanoate with oral or injectable norethisterone preparations. J Clin Endocrinol Metab 87:530–539[Abstract/Free Full Text]
  10. Kinniburgh D, Zhu H, Cheng L, Kicman AT, Baird DT, Anderson RA 2002 Oral desogestrel with testosterone pellets induces consistent suppression of spermatogenesis to azoospermia in both Caucasian and Chinese men. Hum Reprod 17:1490–1501[Abstract/Free Full Text]
  11. Anderson RA, Wallace AM, Wu FC 1996 Comparison between testosterone enanthate-induced azoospermia and oligozoospermia in a male contraceptive study. III. Higher 5{alpha}-reductase activity in oligozoospermic men administered supraphysiological doses of testosterone. J Clin Endocrinol Metab 81:902–908[Abstract]
  12. O’Donnell L, Stanton PG, Wreford NG, Robertson DM, McLachlan RI 1996 Inhibition of 5{alpha}-reductase activity impairs the testosterone-dependent restoration of spermiogenesis in adult rats. Endocrinology 137:2703–2710[Abstract]
  13. Wu FCW, Balasubramanian R, Mulders TMT, Coelingh-Bennink HJT 1999 Oral progestogen combined with testosterone as a potential male contraceptive: additive effects between desogestrel and testosterone enanthate in suppression of spermatogenesis, pituitary-testicular axis, and lipid metabolism. J Clin Endocrinol Metab 84:112–122[Abstract/Free Full Text]
  14. Handelsman DJ, Conway AJ, Howe CJ, Turner L, Mackey MA 1996 Establishing the minimum effective dose and additive effects of depot progestin in suppression of human spermatogenesis by a testosterone depot. J Clin Endocrinal Metab 81:4113–4121[Abstract/Free Full Text]
  15. McLachlan RI, Robertson DM, Pruysers E, Ugoni A, Matsumoto AM, Anawalt BD, Bremner WJ, Meriggiola C 2004 Relationship between serum gonadotropins and spermatogenic suppression in men undergoing steroidal contraceptive treatment. J Clin Endocrinol Metab 89:142–149[Abstract/Free Full Text]
  16. Shah C, Modi D, Sachdeva G, Gadkar S, Puri C 2005 Coexistence of intracellular and membrane-bound progesterone receptors in human testis. J Clin Endocrinol Metab 90:474–483[Abstract/Free Full Text]
  17. El-Hefnawy T, Manna PR, Luconi M, Baldi E, Slotte JP, Huhtaniemi I 2000 Progesterone action in a murine Leydig tumor cell line (mLTC-1), possibly through a nonclassical receptor type. Endocrinology 141:247–255[Abstract/Free Full Text]
  18. Steele EK, Kelly JD, Lewis SE, McNally JA, Sloan JM, McClure N 2000 Testicular sperm extraction by Trucut needle and milking of seminiferous tubules: a technique with high yield and patient acceptability. Fertil Steril 74:380–383[CrossRef][Medline]
  19. Bayne RA, Martins da Silva SJ, Anderson RA 2004 Increased expression of the FIGLA transcription factor is associated with primordial follicle formation in the human fetal ovary. Mol Hum Reprod 10:373–381[Abstract/Free Full Text]
  20. Zhang X, Ding L, Sandford AJ 2005 Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol Biol 6:4
  21. WHO Special Program of Research, Development, and Research Training in Human Reproduction 1999 WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press
  22. Corker CS, Davidson DW 1978 A radioimmunoassay for testosterone in various biological fluids without chromatography. J Steroid Biochem 9:373–374[CrossRef][Medline]
  23. Brady BM, Walton M, Hollow N, Kicman AT, Baird DT, Anderson RA 2004 Depot testosterone with etonogestrel implants result in induction of azoospermia in all men for long-term contraception. Hum Reprod 19:2658–2667[Abstract/Free Full Text]
  24. Baba T, Niida Y, Michikawa Y, Kashiwabara S, Kodaira K, Takenaka M, Kohno N, Gerton GL, Arai Y 1994 An acrosomal protein, sp32, in mammalian sperm is a binding protein specific for two proacrosins and an acrosin intermediate. J Biol Chem 269:10133–10140[Abstract/Free Full Text]
  25. Swerdloff RS, Bagatell CJ, Wang C, Anawalt BD, Berman N, Steiner B, Bremner WJ 1998 Suppression of spermatogenesis in man induced by Nal-Glu gonadotropin releasing hormone antagonist and testosterone enanthate (TE) is maintained by TE alone. J Clin Endocrinol Metab 83:3527–3533[Abstract/Free Full Text]
  26. Matthiesson KL, Amory JK, Berger R, Ugoni A, McLachlan RI, Bremner WJ 2005 Novel male hormonal contraceptive combinations: the hormonal and spermatogenic effects of testosterone and levonorgestrel combined with a 5{alpha}-reductase inhibitor or gonadotropin-releasing hormone antagonist. J Clin Endocrinol Metab 90:91–97[Abstract/Free Full Text]
  27. Simard J, Ricketts ML, Gingras S, Soucy P, Feltus FA, Melner MH 2005 Molecular biology of the 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase gene family. Endocr Rev 26:525–582[Abstract/Free Full Text]
  28. Pratis K, O’Donnell L, Ooi GT, Stanton PG, McLachlan RI, Robertson DM 2003 Differential regulation of rat testicular 5{alpha}-reductase type 1 and 2 isoforms by testosterone and FSH. J Endocrinol 176:393–403[Abstract]
  29. Kinniburgh D, Anderson RA, Baird DT 2000 Suppression of spermatogenesis with desogestrel and testosterone pellets is not enhanced by addition of finasteride. J Androl 22:88–95
  30. McLachlan RI, McDonald J, Rushford D, Robertson DM, Garrett C, Baker HWG 2000 Efficacy and acceptability of testosterone implants, alone or in combination with a 5{alpha}-reductase inhibitor, for male contraception. Contraception 62:73–78[CrossRef][Medline]
  31. McLachlan RI, O’Donnell L, Stanton PG, Balourdos G, Frydenberg M, de Kretser DM, Robertson DM 2002 Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocrinol Metab 87:546–556[Abstract/Free Full Text]
  32. Matthiesson KL, Stanton PG, O’Donnell L, Meachem SJ, Amory JK, Berger R, Bremner WJ, McLachlan RI 2005 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 90:5647–5655[Abstract/Free Full Text]
  33. Mauvais-Jarvis P, Kuttenn F, Baudot N 1974 Inhibition of testosterone conversion to dihydrotestosterone in men treated percutaneously by progesterone. J Clin Endocrinol Metab 38:142–147[Medline]
  34. Cassidenti DL, Paulson RJ, Serafini P, Stanczyk FZ, Lobo RA 1991 Effects of sex steroids on skin 5{alpha}-reductase activity in vitro. Obstet Gynecol 78:103–107[Abstract/Free Full Text]
  35. Rabe T, Kowald A, Ortmann J, Rehberger-Schneider S 2000 Inhibition of skin 5{alpha}-reductase by oral contraceptive progestins in vitro. Gynecol Endocrinol 14:223–230[Medline]
  36. Anderson RA, Irvine DS, Balfour C, Groome NP, Riley SC 1998 Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Reprod 13:920–926[Abstract/Free Full Text]
  37. Andersson AM, Müller J, Skakkebaek NS 1998 Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels. J Clin Endocrinal Metab 83:4451–4458[Abstract/Free Full Text]
  38. Illingworth PJ, Groome NP, Byrd W, Rainey WE, McNeilly AS, Mather JP, Bremner WJ 1996 Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J Clin Endocrinol Metab 81:1321–1325[Abstract]
  39. Anderson RA, Wallace EM, Groome NP, Bellis AJ, Wu FC 1997 Physiological relationships between inhibin B, follicle stimulating hormone secretion and spermatogenesis in normal men and response to gonadotrophin suppression by exogenous testosterone. Hum Reprod 12:746–751[Abstract/Free Full Text]
  40. De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G 2004 A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 101:1327–1332[Abstract/Free Full Text]
  41. Lindsey JS, Wilkinson MF 1996 Pem: a testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis. Dev Biol 179:471–484[CrossRef][Medline]
  42. Takahashi K, Shichijo S, Noguchi M, Hirohata M, Itoh K 1995 Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis. Cancer Res 55:3478–3482[Abstract/Free Full Text]
  43. Steger K, Pauls K, Klonisch T, Franke FE, Bergmann M 2000 Expression of protamine-1 and -2 mRNA during human spermiogenesis. Mol Hum Reprod 6:219–225[Abstract/Free Full Text]
  44. Aoki VW, Carrell DT 2003 Human protamines and the developing spermatid: their structure, function, expression and relationship with male infertility. Asian J Androl 5:315–324[Medline]
  45. O’Donnell L, Narula A, Balourdos G, Gu YQ, Wreford NG, Robertson DM, Bremner WJ, McLachlan RI 2001 Impairment of spermatogonial development and spermiation after testosterone-induced gonadotropin suppression in adult monkeys (Macaca fascicularis). J Clin Endocrinol Metab 86:1814–1822[Abstract/Free Full Text]
  46. Beardsley A, O’Donnell L 2003 Characterization of normal spermiation and spermiation failure induced by hormone suppression in adult rats. Biol Reprod 68:1299–1307[Abstract/Free Full Text]
  47. Nishimura M, Yaguti H, Yoshitsugu H, Naito S, Satoh T 2003 Tissue distribution of mRNA expression of human cytochrome P450 isoforms assessed by high-sensitivity real-time reverse transcription PCR. Yakugaku Zasshi 123:369–375[CrossRef][Medline]
  48. Peng N, Kim JW, Rainey WE, Carr BR, Attia GR 2003 The role of the orphan nuclear receptor, liver receptor homologue-1, in the regulation of human corpus luteum 3ß-hydroxysteroid dehydrogenase type II. J Clin Endocrinol Metab 88:6020–6028[Abstract/Free Full Text]
  49. Kimura Y, Suzuki T, Kaneko C, Darnel AD, Akahira J, Ebina M, Nukiwa T, Sasano H 2003 Expression of androgen receptor and 5{alpha}-reductase types 1 and 2 in early gestation fetal lung: a possible correlation with branching morphogenesis. Clin Sci (Lond) 105:709–713[Medline]
  50. Robinson LL, Gaskell TL, Saunders PT, Anderson RA 2001 Germ cell specific expression of c-kit in the human fetal gonad. Mol Hum Reprod 7:845–852[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J AndrolHome page
R. A. L. Bayne, T. Forster, S. T. G. Burgess, M. Craigon, M. J. Walton, D. T. Baird, P. Ghazal, and R. A. Anderson
Molecular Profiling of the Human Testis Reveals Stringent Pathway-Specific Regulation of RNA Expression Following Gonadotropin Suppression and Progestogen Treatment
J Androl, July 1, 2008; 29(4): 389 - 403.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
S. T. Page, J. K. Amory, and W. J. Bremner
Advances in Male Contraception
Endocr. Rev., June 1, 2008; 29(4): 465 - 493.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
S. T. Page, T. F. Kalhorn, W. J. Bremner, B. D. Anawalt, A. M. Matsumoto, and J. K. Amory
Intratesticular Androgens and Spermatogenesis During Severe Gonadotropin Suppression Induced by Male Hormonal Contraceptive Treatment
J Androl, September 1, 2007; 28(5): 734 - 741.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C. Wang, Y.-G. Cui, X.-H. Wang, Y. Jia, A. Sinha Hikim, Y.-H. Lue, J.-S. Tong, L.-X. Qian, J.-H. Sha, Z.-M. Zhou, et al.
Transient Scrotal Hyperthermia and Levonorgestrel Enhance Testosterone-Induced Spermatogenesis Suppression in Men through Increased Germ Cell Apoptosis
J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3292 - 3304.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/7/2526    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Walton, M. J.
Right arrow Articles by Anderson, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Walton, M. J.
Right arrow Articles by Anderson, R. A.
Related Collections
Right arrow Male Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals