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Chronic Administration of Atrial Natriuretic Peptide Reduces Testosterone Production of Testes in Mice

BI-HUA CHENG

EING-MEI TSAI

CHENG-HUI YANG

YU CHANG

SHIERLEY LI

JAU-NAN LEE

From the ^* School of Medicine, Beijing University, Beijing, China; Department of Obstetrics and Gynecology, Kaohsiung Medical University, Kaohsiung, and Department of Physiology, College of Medicine, National Cheng Kong University, Tainan, Taiwan.

Correspondence to: Dr Jau-Nan Lee, Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd, Kaohsiung, Taiwan (FAX: 886-7-3112493; e-mail: jaunanlee{at}hotmail.com).

Received for publication December 30, 2002; accepted for publication June 4, 2003.

	Abstract

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The purpose of the present study was to examine the effect of the long-term administration of human atrial natriuretic peptide (ANP) on testosterone production in male mice. Twenty-five mice received ANP (20 ng/hour/g body weight) for 7 days via mini-osmotic pump, and the other group (n = 25) received twice-daily intraperitoneal injections. After death, levels of follicle-stimulating hormone, luteinizing hormone (LH), and testosterone in plasma, pituitary gland, and testis were measured by radioimmunoassay. Five mice from each group were examined histologically. In the minipump group, pituitary and plasma levels were significantly higher than those in the control group (771.2 ± 43.6 vs 644.8 ± 24.9 ng/mg and 6.7 ± 0.6 ng/mg vs 2.5 ± 0.6 ng/mL, respectively). In the intraperitoneal group, plasma LH levels were significantly higher in the ANP-treated group than that in control mice (9.6 ± 0.3 ng/mg vs 3.8 ± 0.5 ng/mL), whereas pituitary levels did not differ significantly. In both studies, testicular and plasma testosterone levels were significantly lower than those in control mice (P < .02). Histological features of the testes in ANP-treated mice revealed structural disorganization and inhibition of spermatogenesis. We conclude that the chronic administration of ANP may result in reduced testosterone production due to testicular damage.

     Key words: ANP, spermatogenesis, testosterone

In vitro, ANP can exert direct effects on Leydig cells, stimulating testosterone production (Bex and Corbin, 1985; Mukhopadhyay et al, 1986; Pandey et al, 1986; Schumacher et al, 1993) in a receptor-mediated fashion. ANP also triggers cholesterol side-chain cleavage enzyme (Khurana and Pandey, 1993) via cyclic GMP as a second messenger (Mukhopadhyay et al, 1986), activating cyclic AMP-dependent protein kinase (Schumacher et al, 1992). In vivo, the administration of an ANP bolus (40 ng/g body weight) to rats does not change the secretion activity and morphology of Leydig cells (Mazzocchi et al, 1990). Notably, there is evidence that natriuretic peptides are involved in testicular testosterone production in the fetal rat as early as embryonic day 15.5-19.5, before the onset of pituitary luteinizing hormone (LH) secretion (El-Gehani et al, 2001). In humans, acute alpha-hANP injection increases testosterone concentrations in the spermatic vein significantly, although no difference has been identified in peripheral blood (Foresta et al, 1991). However, most previous studies focused on the acute effects of ANP administration on gonadal cells. There is little information available concerning the long-term effects of ANP on the reproductive system. The present study was designed to examine the effects of long-term ANP administration on testicular testosterone production in mice.

	Materials and Methodsm

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Preparation of Mini-Osmotic ANP-Releasing Pump

hANP (0.5 mg; Nurons Corporation, San Jose, Calif) was dissolved in double-distilled water (500 µL), and 50 µL was added to normal saline (450 µL), to prepare the ANP solution at a concentration of 100 ng/µL. ANP solution (200 µL) was used to fill a mini-osmotic pump (model 2001; Alzet Corporation, Palo Alto, Calif) with a release speed of 2 ng/hour/g body weight. The total amount could be released in 7 days. The ANP solution was injected, using a Hamilton syringe, into the mini-osmotic pump carefully, to avoid gas residue in the pump. All procedures were implemented in an icebox. The pump was then placed into a normal saline bath at 37°C for 4 hours, to reach steady state for ANP release, before it was implanted subcutaneously into mice. Normal saline was used in a similar way in the control group.

Experimental Procedures

One hundred Balb/c male mice (8-9 weeks of age; body weight, 22-27 g) were bred in ad libitum conditions with sufficient water and diet (Purina 5010) in a room at 20°C to 22°C and a light period during 6 AM to 7 PM. The study was designed as follows: group A (n = 25; mean weight ± SD, 24.5 ± 1.5 g) was treated with ANP through a mini-osmotic pump implanted subcutaneously in the neck with a releasing speed of 20 ng/hour/g body weight for 7 days and group B (n = 25; mean weight ± SD, 24.5 ± 2.1 g) received ANP (20 ng/g body weight) twice per day intraperitoneally for 7 days. Another 25 mice in each study were used as control groups. Five mice from each group were selected randomly and examined histologically.

All mice were killed at day 8 by ether anesthesia. Cardio-aspirated blood was obtained immediately after anesthesia. Plasma was separated and stored at -20°C until it was assayed. Fresh tissues, including pituitary glands and whole testis, were weighed and then extracted with a phosphate-buffered saline solution using a high-intensity ultrasonic processor (model VC50; Sonics Materials, Inc, Newton, Conn). After centrifugation (3000 rpm, at 4°C, for 30 min), the supernatant was collected and stored at -20°C until assay. Testes (n = 5) obtained from each of the 4 groups were fixed immediately in formalin solution (35% formaldehyde : water, l : 9) for histological examination. The study was approved by the University Ethical Committee for animal experiments.

Assay Procedures

     LH— LH was determined by a double-antibody radioimmunoassay (RIA), as described by Li (1987), using rat pituitary LH assay kit supplied by the National Hormone and Peptide Program (NHPP), with NIDDK-rLH-RP-3 as the reference standard. The inter- and intra-assay coefficients of variation were 13.1% and 6.3%, respectively. The detection limit of the assay was 0.08 ng per tube.

     Follicle-stimulating hormone— Follicle-stimulating hormone (FSH) was measured by a double-antibody RIA, as described by Li (1993), using a rat pituitary FSH kit supplied by NHPP. The tracer NIDDK-rFSH-I-8 was iodinated using the Iodogen method. The antiserum was NIDDK-anti-rFSH-S-11. The reference preparation was NIDDK-rFSH-RP-2. The inter- and intra-assay coefficients of variation were 12.0% and 8.0%, respectively. The assay detection limit was 0.08 ng per tube.

     Testosterone— Two hundred microliters of standard testosterone (Sigma Chemical Company, St Louis, Mo) or sample was added to 100 µL of anti-testosterone serum (1 : 20 000; kindly supplied by Dr. YL Yu of Academica Sinica, Taiwan) and 100 µL of ³H-labeled (1,2,6,7-³H) testosterone (Amersham Inter Pic, Amersham, United Kingdom) as the tracer. The assay tubes were incubated at 4°C overnight. After the addition of 200 µL of dextran-coated charcoal and centrifugation at 3000 rpm for 20 minutes, the supernatant was removed to another counting tube that contained 2 mL of counting fluid and subjected to Auto beta-ray counter (Packard Tricarb 4000; Packard Instrument Company, Meriden, Conn). Inter- and intra-assay coefficients of variation were 9.0% and 4.6%, respectively. The detection limit of the testosterone assay was 10 pg/tube.

     Histological study— All testes (n = 5 from each group) were obtained from mice at day 7. Tissues were fixed in formalin solution immediately before moving them into paraffin block. Histological examination was done on slides stained with hematoxylin and eosin.

Statistical Analysis

The data presented in RIA measurements are mean ± SD from triplicate determinations. Student's t test was used to test statistical analysis between the study and control groups. P < .05 was defined as the level of statistical significance.

	Results

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In the mini-pump group, LH levels were significantly higher in both pituitary and plasma than in the control group (771.2 ± 43.6 vs 644.8 ± 24.9 ng/mg and 6.7 ± 0.6 vs 2.5 ± 0.6 ng/mL, respectively) (Table 1). Testosterone levels were significantly lower in the testes and plasma from the ANP-treated group than those in control mice (74.4 ± 14.7 vs 234.0 ± 65.0 pg/mg and 136.4 ± 36.9 vs 850.2 ± 136.8 pg/mL, respectively). There was no difference in pituitary gland or plasma FSH levels between study and control groups.

In the intraperitoneal injection group, pituitary LH and FSH levels did not differ significantly between the study and control groups (Table 2). Testis testosterone levels were significantly higher in the ANP-treated group than those in the control group (352.9 ± 21.5 vs 446.1 ± 31.4 pg/mg, respectively). However, plasma testosterone levels were significantly lower in the ANP-treated group than in the control group (903.4 ± 86.8 vs 1504.0 ± 253.2 pg/mL, respectively). Plasma LH levels were significantly higher in the study group than in the control group (9.6 ± 0.3 vs 3.8 ± 0.5 ng/mL).

Histological testing of the testes showed a similar pattern in all ANP-treated mice: the seminiferous tubules were irregularly convoluted and lined by less layered, stratified epithelium (Figure 1). There was a significant reduction in the numbers of cells responsible for spermatogenesis. In addition to the presence of multinucleate cells, spermatids in the luminal space of the study group revealed increased eosinophilic granules in cytoplasm, which denoted degenerative changes. Other features in the interstitial space, including hyperplasia, aggregation of Leydig cells, and loss of integrity of the basement membrane, suggested chemotoxicity.

View larger version (166K): [in this window] [in a new window]   Figure 1. Histological sections of mice testis (5 from each group) prepared by hematoxylin and eosin stain disclose the morphologic differences between (A) the control group and (B to D) study groups. The seminiferous tubules show disorganized and less-stratified epithelium either in the ANP infusion by minipump or intraperitoneal injection groups: (B) apparent increased eosinophilic materials in the luminal space, (C) disrupted spermatogenesis and the narrowed spacing of interstitial tissues, and (D) the appearance of multinucleated giant cells attributed to the possible toxic effects of ANP on luminal sperm. Scale bar indicates (A) 100 µm; (B and C) 50 µm; and (D) 25 µm.

	Discussion

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It has been well documented that, in vitro, ANP stimulates testosterone production in Leydig cells (Mukhopadhyay et al, 1986; Foresta et al, 1991; Schumacher et al, 1992, 1993; Khurana and Pandey, 1993; El-Gehani et al, 2001), which provides evidence of its involvement in reproductive function. However, there is little in vivo information available on the effects of ANP on male gonads. In humans, the acute injection of ANP did not elevate peripheral testosterone and LH levels, but it significantly increased testosterone concentrations in spermatic vein, which suggests a direct influence of ANP on Leydig cells' metabolism (Foresta et al, 1991). In rats, the long-term administration of ANP elevated both basal and hCG-stimulated testosterone levels in blood and caused hypertrophy of Leydig cells (Mazzocchi et al, 1990).

The data obtained in the present study show that the long-term subcutaneous infusion of ANP causes a significant rise in pituitary and plasma LH levels but does not affect FSH levels. Similar results were also seen in the long-term intraperitoneal administration group, although the pituitary LH level did not increase to a statistically significant level. It has been demonstrated that the pituitary gland is not a direct target for ANP, and only very little ANP reaches the brain (Levin et al, 1987). Thus, the raised pituitary LH levels in the long-term subcutaneous infusion group must have been due to a negative-feedback effect of the significant decrease in circulating testosterone levels. On the other hand, in the intraperitoneal group, the insignificant rise in pituitary LH levels was probably the result of less feedback intensity from the lower circulating testosterone levels. The decreased plasma testosterone levels in both groups was apparently attributable to the reduced testicular origin, which was damaged by long-term ANP administration. The testicular histopathologic features agree with the hormonal profiles reached.

However, the long-term administration of ANP (6.5 x 10^-11 M) in mice reduced circulating testosterone levels, which is contrary to the results of a previous report, that long-term ANP infusion resulted in hypertrophy of rat Leydig cells with a consequent increase in testosterone production (Mazzocchi et al, 1990). This discrepancy may be attributed to differences in study design. In the previous study, hCG was administered 60 minutes before the rats were killed. The hCG, then, could have induced testosterone secretion both in vivo (Eisner et al, 2002) and in vitro (El-Gehani et al, 2001) by binding to LH/hCG plasma membrane receptors (Ronco and Llanos, 2000). A recent study that used an antibody raised in adult male rabbits by immunization with a synthetic LH-receptor peptide showed the interaction of endogenous Leydig cell LH receptors with this antibody and explored both hormone agonistic and antagonistic activities (Moudgal et al, 2001). Thus, the blockade of the LH receptor by hCG might affect ANP action on the production of testosterone, especially under long-term administration. ANP was able to cause testosterone production in the rat embryo independent of LH (El-Gehani et al, 2001), which suggests the existence of other mechanisms.

It has been claimed that ANP-specific binding sites are present only in interstitial cells and that other testicular compartments do not undergo significant labeling (Pelletier, 1988). However, an increasing body of evidence has revealed that natriuretic peptide receptors can be found in the spermatids of seminiferous tubules and Leydig cells of the interstitial compartment. The ANP response in vitro is most likely exerted through the pathway of guanylyl cyclase/natriuretic peptide receptor-A (NPRA) and cyclic GMP to produce testosterone in Leydig cells. Guanylyl cyclase activity, the intracellular cyclic GMP level, and ANP receptor density in the testis in vivo, after the long-term administration of ANP, might be key factors that explain the difference between our results and those of in vitro studies. In mice testes, ANP-activated cyclic GMP synthesis provides a good index of NPRA, a receptor guanylate cyclase that synthesizes cyclic GMP in response to ANP. A study of a genetically modified (knockout) mouse uncovered the presence of a novel receptor in testis that preferred BNP over ANP (Goy et al, 2001). Furthermore, immunocytochemically, ANP was expressed in different Leydig cell populations (fetal, progenitor, and immature) with a varied immunoreactive intensity that apparently depended on the acquisition of testosterone producing ability, which suggests a major role in autocrine/paracrine regulation of the rat male gonad (Mourdjeva et al, 2001). The presence of protein kinase-C (PKC) activity in both the seminiferous tubules and Leydig cells demonstrates its involvement in the regulation of these testicular compartments. Its total activity and subcellular distribution varied according to the functional state and endocrine milieu of the testis (Nikula et al, 1987). ANP could negatively regulate phosphorylation of the 78-kDa PKC and the 240-kDa proteins in a cyclic GMP-dependent and -independent manner in cultured Leydig cells (Pandey, 1994). Other evidence also indicates that ANP may act as a negative mediator of "cross-talk" between the PKC and NPRA signaling pathways in murine Leydig cells (Kumar et al, 1997). It has been reported that 4 beta-phorbol-12-myristate 13-acetate, a potent activator of PKC, mediates the inhibition of testosterone production by Leydig cells stimulated with rat atriopeptin II (rAP-II). This inhibition is thought to result from an activation of a phosphodiesterase enzyme, hypothetically through an activated PKC. This leads to a reduction in cellular cyclic GMP content through an increased metabolic removal of cyclic GMP formed in response to rAP-II stimulatiom (Mukhopadhyay and Leidenberger, 1988). Moreover, the dosage of ANP may also affect testosterone production. For instance, at lower ANP concentrations (1 x 10^-11 M), it inhibits testosterone production by LH-stimulated Leydig cells, whereas, at higher concentrations (more than 2 x 10^-9 M) ANP stimulates steroidogenesis beyond the level attained by LH alone (Pandey et al, 1986).

The appearance of numerous multinuclear giant cells formed in the adluminal intratubular compartment after the long-term administration of ANP in the present study mimics the findings of chemotoxic effects on rat testis by ethylene glycol monoethyl ether (Chapin et al, 1984). The histopathologic damage to testicular seminiferous tubules and spermatogenesis clearly indicates the direct action of long-term ANP administration on sperm development and maturation. The mechanism of these changes remains to be evaluated. One possibility is that, for example, the long-term infusion of ANP might, at first, stimulate testicular cells, as was shown in in vitro studies by Mazzocchi et al. (1990). Subsequently, ANP interacts directly with the testicular LH receptors, behaving in a down-regulatory manner, as was shown by our results. The long-term administration of ANP may exert its actions on seminiferous tubules as well as Leydig cells through different signaling pathways other than binding to NPRA to regulate testosterone production, another possibility. It is difficult to measure at present how interactions occur between ANP and hCG in the production of testosterone. Plasma from spermatic veins was not available for our study design, and further studies will be required to see whether the testicular responses are reversible, as are those caused by ethylene glycol monomethyl ether (Aich and Manna, 1996). Indeed, these negative effects, especially those on spermatogenesis, of the long-term administration of ANP may emerge as another issue in male contraception.

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