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Keywords:

  • progesterone receptor;
  • progestin;
  • spermatogenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References

Synthetic progestins such as levonorgestrel (LNG) are used in combination with testosterone (T) in male contraceptive clinical trials to suppress gonadotropins secretion, but whether progestins have additional direct effects on the testis are not known. This study aimed to examine the effect of a potent progestin, (LNG), alone or in combination with testosterone (T) on spermatogenesis in adult rats, and to evaluate the functional role of the progesterone receptors (PRs) in the testis. In comparison with a low dose of LNG treatment in adult rats for 4 weeks, T and T + LNG treatment decreased testicular sperm count to 64.1 and 40.2% of control levels respectively. LNG induced germ cell apoptosis at stages I–IV and XII–XIV; T increased apoptosis at stages VII–VIII; LNG + T treatment induced greater germ cell apoptosis at a wider range of seminiferous epithelial stages. RT-PCR and Western Blots showed that PR was present in testes and up-regulated during suppression of spermatogenesis induced by testicular hormonal deprivation. PR knockout (PRKO) mice had larger testes, greater sperm production, increased numbers of Sertoli and Leydig cells. Suppression of gonadotropin and intratesticular T by GnRH-antagonist treatment induced PR promoter driven LacZ expression in Leydig cells of PRKO mice. This suggests that GnRH-antagonist treatment while inducing germ cell apoptosis also up-regulates PR. We conclude that (i) LNG + T induced greater suppression of spermatogenesis through increase in germ cell apoptosis involving a wider range of seminiferous epithelial stages than either treatment alone, (ii) up-regulation of PR was associated with inhibition of spermatogenesis, (iii) PR knockout mice showed increased sperm production suggesting that testicular PR activated events play a physiological and pharmacological inhibitory role in the testis. These data support the hypothesis that in addition to its known suppressive effects on gonadotropins, progestins may have direct inhibitory actions on the testis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References

The suppression of spermatogenesis by a potential hormonal male contraceptives, testosterone together with a progestin, is achieved through the suppression of LH and FSH, and a decrease in intratesticular testosterone (T). Clinical studies have evaluated the efficacy of spermatogenic suppression with combined regimens based on the administration of an androgen at a dose that maintains physiological peripheral T levels with a second gonadotropin-suppressing agent, such as a progestin (Amory & Bremner, 2003; Wang & Swerdloff, 2004). The addition of a progestin to a regimen of T alone has been shown to promote more rapid and complete spermatogenic suppression (Liu et al.,2008). The combined therapy allows a lower dose of T to be used, thus may reduce potential androgen-related side effects (Meriggiola et al., 2005). While progestins are generally thought to enhance the efficacy of androgens in the suppression of gonadotropin secretion, the underlying mechanisms of the increased efficiency of the combined regimen on spermatogenesis remain unclear.

Progestins and its precursors are produced in large amounts in the testes and are secreted into the blood (Strott et al., 1969). The role of these progestins locally and systemically and the targets of progesterone action in normal male physiology are not known. We hypothesized that progestin acting through progesterone receptors (PRs) may have a direct action on the testis and play an inhibitory role during spermatogenesis. In this study, we examined whether (i) LNG alone, a synthetic progestin, administered as a high dose of 500 mcg/day subcutaneous injections for 6 weeks had any suppressive effects on spermatogenesis in rats, (ii) a lower dose of LNG (3.3 cm implant) would elicit a similar effect, (iii) the addition of this dose of LNG to a sub-optimal dose of T (2 cm implant) would enhance the induction of germ cell apoptosis and the suppression of spermatogenesis in rats compared to that after T alone, (iv) changes in testicular PR occurred at both transcript and protein levels with or without testicular hormonal deprivation. We also studied progesterone receptor knockout mice to characterize their hormonal and testicular phenotypes and to support our hypothesis that progesterone has direct inhibitory actions on spermatogenesis.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References

Animals and experimental protocol

Adult (60-day-old) male Sprague Dawley rats (280–350 g) purchased from Charles River Laboratories, Inc. (Wilmington, MA, USA) were used in the study. PR knockout with LacZ knockin (PRKO) mice, PRKO homozygous, PRKO heterozygous and littermate wild type, were obtained from John Lydon, Ph.D, Baylor College of Medicine. PRKO mice were generated by gene-targeting approaches in which the LacZ reporter encoding β-galactosidase (β-gal) was knocked into exon 1 of the murine PR gene (Ismail et al., 2002). Animals were housed in a standard animal facility under controlled temperature (22 °C) and photoperiod (12 h light, 12 h darkness) with free access to water and rodent chow. Animal handling, experimentation and sacrificing were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Animal Care and Use Review Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center.

Experiment 1. Effect of high dose subcutaneous injection of LNG on suppression of spermatogenesis

To examine the effect of high dose LNG on the male reproductive endocrine axis and spermatogenesis, two groups of four young adult (60-day-old) SD rats received daily subcutaneous injection of either LNG in sesame oil (500 mcg/day) or sesame oil (control) for six consecutive weeks. The rationale for using daily subcutaneously (s.c.) injection of LNG was based on our preliminary experiments which showed that lower doses of LNG (1 cm implant) had no inhibitory actions on gonadotropin secretion in rats. Only when the implant length was increased 10-fold was gonadotropin suppression consistently found (Table 1).

Table 1. Hormone levels in response to various dose of LNG treatment in adult rats
Number of adult rats (n)LNG implant length (cm)Duration of treatment (Weeks)Plasma T (ng/mL)Plasma FSH (ng/mL)Plasma LH (ng/mL)
  1. Values are mean ± SEM.

  2. Means with different superscripts are significantly different (p < 0.05).

40 (Control)61.58 ± 0.19a5.66 ± 0.52a0.93 ± 0.13a
4132.02 ± 1.14a4.73 ± 0.22ab0.78 ± 0.12a
4143.29 ± 1.01a3.92 ± 0.63bc0.55 ± 0.10a
43.342.39 ± 0.06a3.92 ± 0.63bc0.59 ± 0.06a
42040.31 ± 0.09b1.87 ± 0.19d0.16 ± 0.05b
41060.82 ± 0.15c0.35 ± 0.03e0.37 ± 0.04c
32060.76 ± 0.13c1.65 ± 0.49d0.29 ± 0.09bc

Experiment 2. T and/or LNG implant

In previous studies, we showed that 3 cm T silastic implants provided optimal suppression of spermatogenesis in the rat model (Lue et al., 2000). In this experiment, we used 2 cm silastic implant of T to induce sub-optimal suppression of spermatogenesis. The dose of 3.3 cm LNG implant was chosen based on our preliminary dose escalating studies (Table 1) showing that at this dose level, gonadotropins were partially suppressed. Thus, to study the additive or synergistic effect of combined LNG and T treatment, the following experiment was designed. T (2 cm) or LNG (3.3 cm) silastic implants were prepared utilizing polydimethylsilozane tubing (od, 3.18 mm; id, 1.98 mm; Dow Corning Corp., Midland, MI, USA), packed with T or LNG (Sigma, St. Louis, MO, USA), and sealed with silastic medical adhesive A (Dow Corning Corp.) as described (Lue et al., 2000). The release rate of T from the same type of T implant was estimated to be about 30 mcg/cm/day (Zirkin et al., 1989). Under isofluorine anaesthesia, rats were implanted subdermally along the dorsal surface with 2 cm T-filled or 3.3 cm LNG-filled capsules, either alone or in combination of both for 4 weeks. Control rats received the empty implants. To examine the effects of intratesticular hormonal deprivation on PR mRNA expression in testes, groups of four adult (60-day-old) SD rats received a single s.c. injection of water vehicle or GnRH-A (acyline, 30 mg/kg BW) on day 1. Rats were sacrificed on day 6 (Lue et al., 2010).

Experiment 3. Characterization of male reproductive hormonal profile and testicular phenotype of adult PRKO mice

We hypothesized that progestins, mediated by its action on the PR, might play an inhibitory role on spermatogenesis. To test this hypothesis, we examined reproductive hormonal profiles and characterized testicular phenotype of adult (12- to 14-week-old) PR knockout mice by comparing groups of six littermate wild type, five PRKO heterozygous and five PRKO homozygous animals. We examined body weight, testis weight and sperm count in the cauda epididymis, plasma gonadotropin and T levels, as well as morphometric analysis of spermatogenesis. To determine PR localization in the testis, we examined PR promoter driven β-galactosidase (β-gal) expression in groups of four PRKO mice with or without GnRH-A treatment. Knockout mice were treated with GnRH-A [Acyline, 20 mg/kg BW as a single s.c. injection]. After 2 weeks (when germ cell apoptosis would be present) (Lue et al., 2010), the animals were killed and the testes were used for β-gal staining.

Blood collection and tissue preparation

Both control and experimental animals were injected with heparin (130 IU/100 g BW, i.p.) 15 min before being sacrificed by a lethal injection of sodium pentobarbital (100 mg/kg BW i.p.) to facilitate testicular perfusion using a whole body perfusion technique (SinhaHikim & Swerdloff, 1993). Body weight was recorded at autopsy. Blood samples were collected from the right ventricle of each animal immediately after death, and plasma was separated and stored at −20 °C for subsequent hormone assays. Before perfusion, one testis from each animal was removed, weighed and after decapsulation the testicular parenchyma was either snap-frozen in liquid nitrogen for RNA and protein extraction or used for determining the number of advanced (step 18–19) spermatids by the homogenization technique. Then, one side of cauda epididymis from each mouse was dissected out and minced in PBS (pH 7.4) for sperm count (Lue et al., 1998). The contralateral testes were then fixed by vascular perfusion with either 5% glutaraldehyde in 0.05 m cacodylate buffer (pH 7.4) or Bouin's solution for 30 min, preceded by a brief saline wash. The testes were removed, cut into small transverse slices and placed into the same fixative overnight. One slice from the middle region of fixed testis was processed for routine paraffin embedding for histological evaluation and in situ detection of apoptosis.

Hormone assays

Testosterone concentrations in plasma were measured using RIA as reported (Lue et al., 2000). The minimal detection limit in the assay was 0.25 ng/mL; the intra- and inter-assay coefficients of variations were 8 and 11% respectively. Plasma LH levels were measured by modified immunofluorometric assay (IFMA) as described (Haavisto et al., 1993). The minimal detection limit of the assay was 0.02 ng/mL. The intra- and inter-assay coefficients of variation were 6.9 and 12.3% respectively. Plasma FSH levels were measured by immunofluorometric assay (Lue et al., 2000) using reagents supplied by Organon (OSS, the Netherlands). The minimal detection limit in the assay was 0.04 ng/mL. The intra- and inter-assay coefficients of variations were 4.7 and 6.0% respectively.

Assessment of apoptosis

In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labelling (TUNEL) technique using an Apop Tag-peroxidase kit (Intergen Co, Purchase, NY, USA) as described earlier (Lue et al., 2000). Negative and positive controls were carried out in every assay. As negative controls, tissue sections were processed in an identical manner, except that the TdT enzyme was substituted with the same volume of PBS. Testicular sections from rats treated with 3 cm testosterone implant for 14 days were used as positive controls. Enumeration of the viable Sertoli nuclei with distinct nucleoli and apoptotic germ cell population was carried out at stages I–IV, V–VI, VII–VIII, IX–XI and XII–XIV using an Olympus BH-2 microscope with an X100 oil immersion objective (Olympus, Tokyo, Japan). For each testis, at least 10 tubules per stage group were used. These stages were identified according to the criteria proposed by Russell et al. (Russell et al., 1990) for paraffin sections. The rate of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per 100 Sertoli cells (Lue et al., 2000).

Western blot analysis

Protein extraction and Western blotting were performed as described (Lue et al., 2002). Briefly, 80 μg of protein was resolved on a 7.5% SDS polyacrylamide gel at 160 V in a Mini-Protean II Cell (Bio-Rad Laboratories, Inc. Hercules, CA, USA). Equal loading was examined by running a separate gel in parallel and staining with Coomassie blue and demonstrated by actin expression on the same gel. Proteins were transferred to 0.45 μm nitrocellulose membranes in transfer buffer (25 mm Tris-base, 190 mm glycine, 20% methanol) at 100 V for 1 h in the cold. Efficiency of transfer was determined using Ponceau S (Sigma). Membranes were blocked in 5% non-fat dried milk in TTBS (0.9% NaCl, 0.1% Tween 20, 100 mm Tris-HCl, pH 7.5) and then incubated with the primary antibody [1 : 200 PR (Code no. A0098; DAKO Corporation, Carpinteria, Denmark)] (Kurita et al., 2001) at 4 °C overnight. Following three 10-min washes in TTBS, membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Life Science Inc. Arlington Heights, IL, USA) secondary antibodies at a 1 : 2000 dilution. For immunodetection, membranes were incubated with SuperSignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL, USA) and exposed to Fuji X-ray film (Fuji Medical Systems, Inc., Stamford, CT, USA). To confirm the specificity of the PR antibody, protein extracts of rat uterus with or without primary antibody were used as positive controls. In addition, testicular lysates from PRKO mice were used as negative control for further testing the antibody specificity (Mote et al., 2006).

RNA isolation and real-time RT-PCR procedure

RNA was isolated from each rat testis, respectively, using The RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). Briefly, testis parenchyma preserved in RNAlater (Ambion, Austin, TX, USA) was homogenized in a denaturing buffer. The lysates were then passed through a gDNA Eliminator spin column. Ethanol was added to the flow-through, and the samples were applied to an RNeasy spin column. RNA binding to the membrane was eluted in water. RNA concentration was measured with NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). A one-step real-time RT-PCR was performed using QuantiTect SYBR Green RT-PCR kit (Qiagen) in a 96-well plate using a 7000 ABI prism sequence detection system (Applied Biosystems, Foster City, CA, USA). About 100 ng of total RNA from each rat testis was plated in triplicate RT-PCR reactions with PR primers (PR-B F: 5′-CCAATACCGATCTCCCTGGAC-3′; R: 5′-CTTCCACTCCAGAGAAAGCTCC-3′) and the raw values normalized to a housekeeping gene beta-Actin (Beta Actin F: 5′-TCATGAAGTGTGACGTTGACATCCGT-3′; R: 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′) (Turgeon & Waring, 2006). The quantification of the gene expression fold change was calculated by using the formula 2−∆CT.

β-galactosidase staining procedure

Testes harvested from PRKO mice treated with or without GnRH-A for 2 weeks were subjected to β-gal staining using LacZ Tissue Staining Kit (InvivoGen, San Diego, CA, USA). Briefly, decapsulated testis parenchyma was fixed in fixative solution on ice for 1 h. After washing with PBS, tissues were incubated with staining solution containing X-gal (5-bromo-4-choro-3-3indoyl-β-d-Galactopyranoside) at 37 °C for 4–5 h in the dark. After five times of PBS washing, testes were immersed into 70% ethanol for subsequent cryosections. The testicular sections were counterstained with Nuclear Fast Red Solution (ScyTek Laboratories, Inc., Logan, UT, USA).

Morphometric procedures

The volume densities (Vv) of seminiferous tubules, tubular lumens, interstitium and Leydig cells in PRKO homozygous, heterozygous and their littermate controls were determined by point-counting method (SinhaHikim & Swerdloff, 1993, 1994). Numerical densities (Nv) of Sertoli and germ cells (number per unit volume of the seminiferous tubule) at stage VII–VIII of the cycle in PRKO homozygous, heterozygous and their littermate controls were determined by stereological techniques as described (SinhaHikim & Swerdloff, 1995; Lue et al., 2003). The Leydig cell volume and the number were determined, employing glutaraldehyde-fixed, epoxy-embedded, toluidine blue-stained testicular sections, by accepted stereological technique, as described (Lue et al., 2001; Yamamoto et al., 2001). A comparison between the results of stereological analyses of Leydig cell number obtained via the unbiased disector method and the present method demonstrated that the assumption of nearly spherical shape of the Leydig cell nuclei in non-regressed testis had no significant effect on the estimation of Leydig cell number (Mendis-Handagama & Ewing, 1990).

Statistical analysis

Statistical analyses were performed using the Sigmastat 2.0 Program (Jandel Cooperation, San Rafael, CA, USA). Results were tested for statistical significance using t-test for comparing data between LNG treated alone and control groups in rats. Student-Newman-Keuls Method tests after one-way anova was used for comparing data among control, LNG alone, T alone and T + LNG groups in rats, and among PRKO homozygous, heterozygous and their littermate control mice. Differences were considered significant if p < 0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References

High dose LNG suppresses serum LH, FSH, T and intratesticular T, and induces stage-specific activation of germ cell apoptosis

Body and testis weights and plasma hormone levels in control and high dose of LNG-treated rats are summarized in Table 2. After 6 weeks of a high dose of LNG subcutaneous administration (500 mcg/day), there were no changes in body weight. Testis weight in LNG-treated group was significantly decreased compared with controls (p < 0.05). Plasma FSH, LH and T and intratesticular T levels were markedly suppressed by LNG administration. Activation of germ cell apoptosis after a high dose of LNG treatment was present in stages I–IV, VII–VIII and XII–XIV, and the changes in the incidence of apoptosis (expressed as numbers/100 Sertoli cells) are summarized in Fig. 1. As compared with controls, the incidence of germ cell apoptosis at stages I–IV, VII–VIII and XII–XIV was significantly increased after LNG treatment (p < 0.05).

Table 2. Organ weight and hormone levels 6 weeks after a high dose (500 mcg/day) of LNG treatment in rats
ParameterControl (n = 4)LNG (n = 4)
  1. Values are mean ± SEM.

  2. a

    Significant difference p < 0.05.

Body weight (g)498.30 ± 25.50512.30 ± 23.10
Testis weight (g)1.86 ± 0.191.06 ± 0.12a
Plasma T (ng/mL)3.81 ± 1.410.05 ± 0.01a
Plasma FSH (ng/mL)6.23 ± 1.420.83 ± 0.16a
Plasma LH (ng/mL)1.12 ± 0.260.02 ± 0.01a
image

Figure 1. In situ detection of apoptosis by TUNEL assay. Compared to the control group (A), s.c. LNG administered to rats at high dose (500 μg/day) for 6 weeks resulted in increased germ cell apoptosis at stage VII (B), stage III (C) and stage XIV (D). Apoptotic germ cells are shown as dark brown in colour (Magnification, X250; Scale bar, 0.05 mm). Quantitative assessment of germ cell apoptosis at various stages of seminiferous epithelium cycles shows that the rate of germ cell apoptosis was significantly increased (*< 0.05) at early (I–IV), middle (VII–VIII) and late (XII–XIV) stages of the seminiferous epithelial cycle. All values are expressed as mean ± SEM.

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Combination of T and LNG results in a rapid suppression of spermatogenesis

Testis weight and testicular sperm count in rats were not decreased in the LNG (3.3 cm) alone treated group, but significantly decreased in T-treated group compared with either control or LNG alone group. The combination of LNG and T further significantly decreased testis weight and sperm count than either treatment alone. Plasma T levels were maintained in all groups. Plasma LH and intratesticular T levels were not decreased in the LNG alone group, but were suppressed equally in both T alone and T + LNG groups. Plasma FSH levels were significantly decreased in LNG alone group as compared with controls, but the FSH concentrations were even more suppressed in the T and T + LNG groups (Fig. 2).

image

Figure 2. Body weight, testis weight and testicular sperm count from control, LNG (3.3 cm) alone, T (2 cm) alone and T in combination with LNG (T + LNG)-treated groups. Plasma FSH, LH and intratesticular T levels from control, LNG (3.3 cm) alone, T (2 cm) alone and T in combination with LNG (T + LNG)-treated groups. All values are the mean ± SEM. Means with different superscripts are significantly different (p < 0.05).

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In contrast to the increase in germ cell apoptosis at androgen-dependent middle stages VII–VIII in the T-treated group, LNG alone induced activation of apoptosis exclusively at early stages I–IV and late stages XII–XIV. In the T + LNG group, in addition to increase in apoptosis at stages I–IV and XII–XIV as compared with control, a further significant increase in apoptosis was also noted at stages V–VI, VII–VIII and IX–XI (Table 3). Sperm retention was observed at stages VII–XI in T alone group, but not in LNG alone and control groups. Addition of LNG to T further extended sperm retention to stages VII–XI. The spermatozoa that failed to release from seminiferous tubules underwent apoptosis and were located near the basal lamina (Fig. 3).

Table 3. Quantitative assessment of germ cell apoptosis at various stages of seminiferous epithelial cycles in adult rats
Groups (n = 4/group)I–IVV–VIVII–VIIIIX–XIXII–XIV
  1. Values are mean ± SEM of the rate of germ cell apoptosis (apoptotic index) expressed as the number of apoptotic germ cells per 100 Sertoli cells.

  2. Means with different superscripts are significantly different (p < 0.05).

Control4.59 ± 0.92a0.02 ± 0.01a0.02 ± 0.01a3.18 ± 1.09a8.98 ± 1.29a
LNG implant (3.3 cm)19.33 ± 1.52b0.02 ± 0.01a0.02 ± 0.01a2.16 ± 0.59a14.87 ± 0.49b
T implant (2 cm)4.23 ± 0.42a0.29 ± 0.27b32.81 ± 2.95b1.63 ± 0.29a8.38 ± 0.62a
T + LNG impants19.34 ± 1.85b5.01 ± 0.53c52.87 ± 3.12c25.28 ± 2.62b15.16 ± 0.61b
image

Figure 3. Compared to control (A), LNG or T implant alone, T+LNG (B) induced greater retention of mature spermatids. Note that the spermatozoa that failed to release from seminiferous tubules underwent apoptosis and were located near the basal lamina (TUNEL positive staining shown in dark brown in colour). Magnification: Scale bar, 0.02 mm.

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The effect of hormone perturbation on PR expression in the testis

To address the question of whether and where PRs were localized in the testis, we examined PR promoter driven β-gal expression in PRKO mice with and without GnRH-A treatment (GnRH-A suppressed intratesticular T and induced germ cell apoptosis). (Sinha Hikim et al., 1997). We found little or no blue staining (indicating β-gal expression) in Leydig cells of untreated PRKO mice. In contrast, a prominent blue staining was found in Leydig cells of PRKO mice treated with GnRH-A for 2 weeks (Fig. 4). These results demonstrated that PR was localized in Leydig cells and PR promoter driven LacZ expression could be induced by intratesticular hormonal deprivation. To determine if suppression of spermatogenesis had any effect on PR expression in the testis, we also performed Real-time PR-PCR and Western Blot analysis. Our results showed that PR-B was present in rat testes at both transcript and protein levels. The PR-B receptor was up-regulated in T alone; LNG alone and T + LNG-treated animals as compared with controls (Fig. 5). Real-time RT-PCR for PR-B showed that PR-B was 1.89-fold increased in testes of rats treated with GnRH-A for 5 days when intratesticular testosterone was decreased. This result was consistent with our microarray data showing PR-B was increased by 3.2-fold and 2.42-fold in testes of rats treated with 3 cm T implant for 1 and 2 weeks, respectively, as compared with controls (Lue et al., 2004). Taken together, the results showed that intratesticular hormonal deprivation up-regulated PR-B expression in testes associated with the suppression of spermatogenesis. The 60 kDa bands, which parallel the molecular weight corresponding to PR-C or progesterone-binding protein (Peluso & Pappalardo, 1998), were found by Western blot (data not shown) to be present with similar expression in controls, and testes treated with LNG, T or T + LNG for 4 weeks in rats.

image

Figure 4. Representative light micrographs show a faint blue staining (β-gal) in the Leydig cells of PRKO mice (A). A prominent blue staining can be visualized in Leydig cells (red arrows) of PRKO mice 2 weeks after a single s.c. injection of GnRH-A to suppress intratesticular T levels (B). Magnification: Scale bar, 0.05 mm.

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image

Figure 5. Western blot shows the progesterone receptor from control (C), T alone (T), LNG alone and LNG + T-treated rat testes. In control testes, there were faint PR-B bands present. After 4 weeks of treatment with T, LNG or T + LNG, PR-B (arrow) was up-regulated in the whole testicular lysates. The actin bands detected from the same membrane demonstrated equal loading. The densitometry data of the ratio of PR-B over actin exhibits the markedly increased PR-B expression after intratesticular hormonal deprivation as compared with controls.

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Characterization of reproductive hormonal profile and testicular phenotype of PRKO mice

We examined reproductive hormonal profiles and characterized testicular phenotype of PRKO homozygous as compared with littermate wild type, and PRKO heterozygous mice. PR knockout mice had larger testes and significantly higher sperm count in the cauda epididymis as compared with wild type controls, but not with heterozygous mice. Plasma LH, T and LH to T levels were similar in all three groups of animals, but FSH was significantly decreased in PRKO homozygous as compared with wild type, but not with the heterozygous mice (Table 4). Morphometric analysis showed (i) significant increase in the absolute volume of seminiferous epithelium (VSE) in homozygous (VSE = 104.89 ± 5.37 μL) as compared with wild type (VSE = 81.46 ± 5.49 μL, p < 0.05), but not with heterozygous males (VSE = 94.59 ± 6.35 μL), (ii) significant increase in the number of Sertoli cells and Leydig cells in heterozygous more evident in homozygous as compared with wild type mice (Table 5), and (iii) no difference in the ratio of spermatogonia, preleptotene and pachytene spermatocytes, and round spermatids per Sertoli cell among PRKO homozygous, heterozygous and their littermate wild type males (Table 5).

Table 4. Body and testis weights and plasma hormone levels in PR knockout mice
ParameterWildtype (n = 6)Heterozygous (n = 5)Homozygous (n = 5)
  1. Values are mean ± SEM.

  2. Means with different superscripts are significantly different (p < 0.05).

Body weight (g)30.90 ± 1.3730.98 ± 5.0531.32 ± 3.63
Testis weight (mg)107 ± 8.53a119 ± 13.1ab126 ± 12.6b
Sperm count in the cauda epididymis (106/cauda)5.28 ± 0.13a5.16 ± 0.22ab6.17 ± 0.36b
LH (ng/mL)0.13 ± 0.090.10 ± 0.050.36 ± 0.32
FSH (ng/mL)32.86 ± 3.41a24.91 ± 3.70ab21.09 ± 1.06b
T (ng/mL)1.80 ± 0.861.92 ± 0.931.07 ± 0.46
LH/T0.10 ± 0.020.16 ± 0.060.18 ± 0.10
Table 5. Comparison of Sertoli cell number and germ cell to Sertoli cell ratios at stages VII-VIII in wildtype and PR knockout mice
Cell typesWildtype (n = 4) Heterozygous (n = 4)Homozygous (= 4)
  1. Values are mean ± SEM.

  2. Means with different superscripts are significantly different (p < 0.05).

Sertoli cells (106/testis)2.37 ± 0.19a3.02 ± 0.11b3.80 ± 0.23c
Leydig cells (106/testis)0.74 ± 0.02a1.56 ± 0.25b1.80 ± 0.16c
Germ cell to Sertoli cell ratios at stages VII–VIII
Spermatogonia0.13 ± 0.030.12 ± 0.010.14 ± 0.02
Preleptotene spermatocytes4.63 ± 0.134.49 ± 0.234.07 ± 0.53
Pachytene spermatocytes4.68 ± 0.044.65 ± 0.294.58 ± 0.67
Round spermatids11.16 ± 1.0811.37 ± 0.159.73 ± 1.15

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References

To investigate the action of progestins in the testis, we chose to use LNG, a progestin studied in many human male contraceptive trials and we utilized a rat model which had been shown in prior studies to reflect similar changes in men when exposed to exogenous T and progestins. We first demonstrated that a high dose of LNG (500 mcg/day s.c. for six consecutive weeks) suppressed gonadotropin secretion, decreased intratesticular T levels and induced stage-specific activation of germ cell apoptosis. The increased apoptosis was not only found at the expected androgen-sensitive middle stages (VII–VIII), but also at early (I–IV) and late (XII–XIV) stages of the seminiferous epithelial cycle. Our earlier studies showed that a decrease in intratesticular T induced by T administration alone (3 cm T implant) increased apoptosis exclusively at stages VII–VIII (Lue et al., 2000). Thus, the results from these studies suggest that in addition to suppression of gonadotropins, LNG may have a direct action on spermatogenesis. When sub-optimal dose of T implants were combined with a lower dose of LNG that on its own had inadequate efficacy, gonadotropins and intratesticular T levels were suppressed to the same degree as induced by optimal dose of T alone. More suppression of spermatogenesis occurred in the combined T + LNG group through greater increase in apoptosis and more retention of apoptotic mature spermatids. This additive action of LNG on T implants is similar to the observations in men (Anawalt et al., 1999; Liu et al., 2008), and is mostly ascribed to additive suppression of the two steroids on FSH and LH secretion. It should be noted that the dose of LNG used in the current study in rodents are many fold higher than the dose of this progestin used for spermatogenesis suppression in men. The reason why such high doses of progestins are required in rodents is not clear, but may be related to the absence of steroid-binding proteins in the sera of rodents and the rapid clearance of progestins such as LNG from the body. It is interesting to note that a low dose of LNG implant (3.3 cm) alone for 4 weeks selectively suppressed FSH secretion without noticeable effects on LH and T and increased germ cell apoptosis at early (I–IV) and late (XII–XIV) stages of spermatogenic epithelial cycles in rats. This is consistent with the results from FSH immuno-neutralization experiments reported by Meachem et al. (Meachem et al., 1999).

The physiological significance of progestins as paracrine or endocrine modulators of testicular function remains an interesting and under explored question. Experimental studies have suggested the possibility of a direct inhibitory action of progestins at the gonadal level. Medroxyprogesterone acetate treatment in rats resulted in a lowering of plasma levels of T, androstenedione and LH, as well as a reduction of epididymal sperm counts. The progestin-treated groups showed markedly lower levels of testicular 17β-hydroxy-steroid dehydrogenase activity (Fotherby et al., 1972; Satyawaroop & Gurpide, 1978). Progestin-protein interactions have been observed in testicular cells (Schmidt & Danzo, 1980; Wagle et al., 1983; El-Hefnawy et al., 2000) suggesting the possibility of a direct role of progesterone in the regulation of male reproduction. The tritiated progestin R5020 showed high affinity binding to charcoal-treated cytosolic fractions prepared from purified Leydig cells (Pino & Valladares, 1988). Progestins have been reported to have inhibitory effects on the testis including 5α-reductase inhibitory activity and interference with androgen biosynthesis and action (Mauvais-Jarvis et al., 1974). They exert rapid non-genomic effects on Leydig cells, spermatozoa and affect sperm function (Lishko et al., 2011; Strunker et al., 2011) such as motility, capacitation, acrosome reaction and the ability to bind to zona proteins (Baldi et al., 1995, 1999). Some progestins, like LNG, also bind to androgen receptors (Phillips et al., 1990; Kumar et al., 2000) and exert androgenic activities such as decreasing blood sex hormone binding globulin and HDL-cholesterol levels. Clinical studies showed that combining progestins with androgens lead to an enhanced suppression of gonadotropins and spermatogenesis by inhibiting spermatogonial proliferation and spermiation in the human testes (McLachlan et al., 2002). We and others have demonstrated that addition of progestins to T induced greater alteration of testicular gene expression than T treatment alone in men (Walton et al., 2006; Lue et al., 2008). In this study, although T alone and T + LNG suppressed gonadotropin (LH and FSH) secretion equally, T + LNG induces a greater suppression on spermatogenesis than T treatment alone. Thus, these data support the hypothesis that in addition to suppression of gonadotropins, progestins may have a direct action on testes.

The physiological effects of progesterone are primarily mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors (Giangrande & McDonnell, 1999). Receptors for progesterone are expressed at least as two isoforms, PR-A and PR-B in the nucleus. PR-A, is a truncated form of PR-B, lacking the B upstream sequence (amino acids 1 to 164). While the two forms of PR have similar DNA- and ligand-binding affinities, they have opposite transcriptional activities (Conneely & Lydon, 2000). Different tissues may express different proportions of PR-A and PR-B (Conneely et al., 2002). Our data demonstrated that up-regulation of PR-B protein levels in the testis was associated with the suppression of spermatogenesis induced by intratesticular hormonal deprivation. This inverse relationship between the suppression of spermatogenesis and the increased PR-B protein levels suggests that PR present in the testis may play an inhibitory role in spermatogenesis. In addition to these two well-established PR isoforms PR-A and PR-B, evidence was presented for a third human PR, and N-terminally truncated, termed the C-receptor (Wei et al., 1990, 1996; Wei & Miner, 1994). PR-C lacks the N-terminus and the first DNA-binding finger of PR, but contains the second DNA-binding finger, the hinge region and the hormone-binding domain. We found 60 kDa bands, which parallel the molecular weight corresponding to PR-C or progesterone binding protein (Peluso & Pappalardo, 1998), predominantly expressed in the testis. We did not find apparent changes of this protein after hormonal deprivation. The sites of PR localization in the testis are controversial. PR localization detected by immunohistochemistry was quite variable in testes depending on antibodies used. PR mRNA and protein have been detected in nucleus and cytoplasm of spermatogonia, spermatocytes and round spermatids, Sertoli cells and the Leydig cells in human testes (Shah et al., 2005; Han et al., 2009). PR have been predominantly localized in pre-spermatogonia, A and B spermatogonia, peritubular myoid cells of both immature and mature boar testis (Kohler et al., 2007). Another study reported that PR was expressed in a few peritubular and interstitial cells, but not in germ cells of human and non-human primates (Luetjens et al., 2006). PR has been shown to be present in rodent Leydig cells (Pino & Valladares, 1988; El-Hefnawy et al., 2000; Gonzalez et al., 2010). In this study, we examined the PR localization in mouse testes by detecting PR promoter driven β-gal expression, and demonstrated that intratesticular hormonal deprivation resulted in PR promoter driven LacZ expression predominantly in Leydig cells. This finding suggests that the direct action of progestins on the testis may be mediated through PR in Leydig cells to modulate Leydig cell functions that in turn may affect spermatogenesis.

To further decipher the physiological roles played by PR in the male, as well as to study directly PR function in an in vivo context, PR knockout (PRKO) mice were generated (Lydon et al., 1995, 1996; Schneider et al., 2005). Both male PRKO mice have lower FSH levels, higher inhibin levels and similar serum LH, testosterone, progesterone levels when compared to wild type mice. Testis weight and morphology are grossly normal. Young male mice had increased sperm concentration in the vas deferens. PRKO males exhibit significant enhancement in sexual behaviour (Schneider et al., 2005). In this study, we characterized the plasma hormonal profile and testicular phenotype in PRKO mice by comparing testicular phenotype of PRKO homozygous, heterozygous and their littermate wild type male mice, which eliminated the variations caused by different genetic strains in mice. We found PR knockout mice had larger testes and significantly increased sperm count in the cauda epididymis as compared with wild type, but not with heterozygous mice. Plasma LH and T levels were similar in all three groups of animals, but FSH was significantly decreased in homozygous as compared with wild type, but not with heterozygous mice. Using morphometric analysis established in our laboratory, we demonstrated that PR knockout mice have more Leydig and Sertoli cell numbers with increased sperm number output. The optical disector technique has been used to quantify the number of germ cells especially in regressed testis (McLachlan et al., 1995). In this study, we used the glutaraldehyde perfused and fixed, epoxy-embedded, toluidine blue-stained thin sections (1 μm in thickness) for morphometric analysis. The epoxy-embedded testis samples were well preserved and significantly reduced the shrinkage factor as compared with the paraffin-embedded tissues to eliminate quantification errors. We chose to adopt an accepted stereological method that had been employed in studies by us as well as by others to determine the Leydig cell volume and the number (SinhaHikim et al., 1991; France et al., 2000; Lue et al., 2001; Yamamoto et al., 2001). A comparison between the results of stereological analyses of Leydig cell number obtained via the unbiased disector method and the present method further demonstrated that the assumption of nearly spherical shape of the Leydig cell nuclei in non-regressed testis had no significant effect on the estimation of Leydig cell number (Mendis-Handagama & Ewing, 1990). However, the results can be flawed if the same assumption is made for atrophied Leydig cell nuclei with irregular profile. This problem, however, does not arise for our study as we used stereological method to quantify wild type, heterozygous and homozygous of PRKO mice, which exhibit normal testicular histology. Our results suggest that progestins may play an inhibitory role in the spermatogenesis mediated by PR in the testis. Progestins may also play an inhibitory role to limit the number of Leydig cells and Sertoli cells during development and in turn regulate germ cell proliferation and meiosis during spermatogenesis. The absence of PR unleashes this inhibitory effect resulting in more Sertoli cell and consequent spermatozoa, further suggesting a direct effect of progestins on the testis.

In summary, we demonstrated that (i) higher dose of LNG suppresses gonadotropin secretion, decreases intratesticular T and increases germ cell apoptosis, (ii) lower dose of LNG alone selectively suppresses FSH resulting in activation of germ cell apoptosis at stages I–IV and XII–XIV, but not in the androgen-sensitive stages, (iii) T induces germ cell apoptosis exclusively at stages VII–VIII and causes some degree of mature spermatids retention, (iv) combination of T and LNG suppresses gonadotropins and intratesticular T levels and induces greater suppression of spermatogenesis through accelerated germ cell apoptosis involving a wider range of stages and more retention of apoptotic mature spermatids than T or LNG alone, (v) up-regulation of PR is associated with suppression of spermatogenesis induced by hormonal deprivation, (vi) PRKO mice have larger testes, more sperm production, increased numbers of Sertoli cells and Leydig cells as compared with their littermate heterozygous and wild type controls. These findings together suggest that progestin action mediated through PR may play physiological and pharmacological inhibitory roles in the testis. These finding may also have clinical implications to unravel the mechanisms of why addition of progestins to T results in a more rapid and complete suppression of spermatogenesis (Liu et al., 2008). It is probable that in addition to suppression of gonadotropins, progestins directly acts on the PR in Leydig cells to induce more complete testicular hormonal deprivation leading to accelerated germ cell apoptosis during spermatogenesis. In the absence of PR, the direct action of progestins on the testis is eliminated leading to functionally more active spermatogenesis resulting in increased sperm production.

Author's Contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References

YanHe Lue designed and performed the research, analysed the data and wrote the manuscript. Christina Wang and Ronald Swerdloff were deeply involved in study design, reviewed the data and revised the manuscript. John Lydon contributed the PR knockout mice and revised the manuscript. Andrew Leung performed hormone analysis and James Li performed LacZ staining and tissue preparation.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. Author's Contributions
  9. References
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