Mutant phenotype analysis suggests potential roles for C-type natriuretic peptide receptor (NPR-B) in male mouse fertility

Background C-type natriuretic peptide (CNP) signaling through its receptor natriuretic peptide receptor B (NPR-B) is a key molecule for mammalian reproduction, and known to play important roles in female fertility. However, the function of these peptides in mouse male reproduction remains largely unknown. To determine the role of CNP/NPR-B signaling in male reproduction we investigated phenotype of Npr2-deficient short-limbed-dwarfism (Npr2 slw/slw ) mice, which have been shown to have gastrointestinal (GI) abnormalities. Findings In homozygous Npr2 slw/slw mice, spermatogenesis is developmentally delayed at both 2 and 4 weeks of age, with vacuolation and degenerating apoptotic germ cells being observed at 3 weeks age. However, the adult Npr2 slw/slw mice exhibited apparently normal spermatogenesis, albeit with some aberrant spermatids, suggesting that developmental delay was overcome. In addition, the adult Npr2 slw/slw mice showed abnormal penile morphology (paraphimosis). Conclusions The potential role of CNP signaling via the NPR-B receptor in male fertility appears to be mediated not through germ-cell development, but may be through maintenance of normal penile function.


Background
The mouse Npr2 gene encodes natriuretic peptide receptor B, NPR-B (also known as NPR2), which is a known receptor for C-type natriuretic peptide, CNP (encoded by the Nppc gene, and also known as NPPC). NPR-B synthesizes intracellular cyclic guanosine monophosphate (cGMP) [1], which acts as a secondary messenger and is degraded by cGMP-specific phosphodiesterase type 5 (PDE5) [2]. The peptide and its receptor, CNP/NPR-B are expressed in the nervous and vascular systems [3][4][5], including the gastrointestinal tract [6], where they are thought to act as a local paracrine or autocrine regulators [5].
A number of reports have demonstrated an important role for CNP in reproductive regulation in females [7].
Although male Npr2 exhibit reduced reproductive functions, it is not known why male Npr2 mutants are infertile or sub-fertile. Most spontaneous Npr2 slw/slw (SLW: Shortlimbed dwarfism) males fail to mate, but a limited number of these mutants generated offspring [8]. Males homozygous for another spontaneous mutation, Npr2 pwe/pwe (peewee mouse), were sub-fertile, but exhibited normal testis histology [9]. Interestingly, the male Npr2-KO mouse was infertile but exhibited normal spermatogenesis [10]. Observations on male reproduction were mentioned only briefly in these studies and not pursued in detail.
In a previous report, it was suggested that CNP/NPR-B signaling could lead to relaxation of seminiferous tubules and thereby regulate sperm transportation and testicular blood supply [11]. This signaling is also believed to modulate spermatozoa motility, testicular germ cell development and testosterone synthesis in mice [12]. In addition, it was also reported that CNP regulates blood-testis barrier (BTB) dynamics in adult rats [13].
These reports suggested that CNP is a key factor for spermatogenesis. Therefore, it might be expected that absence of NPR-B would also impact on spermatogenesis, and indeed, reduced fertility is a phenotype in several Npr2 mutants. However, the Npr2 mutant mice also exhibit other phenotypes that might ultimately affect fertility. For example, Npr2 slw/slw mutants display gastrointestinal (GI) dysfunctions and high pre-weaning death, due to lack of CNP/NPR-B signaling [6,14]. In the case of the few surviving Npr2 slw/slw mice, the GI disorders might cause nutritional restriction, which is known to contribute to aberrant spermatogenesis and reduced number of germ cells [15,16].
This study was undertaken to provide more complete and definitive observation using mice homozygous for a mutant allele of Npr2, Npr2 slw , which could contribute to determination of a possible link between the CNP/ NPR-B signaling pathway and male reproduction.

Mice
The Npr2 slw mutant line, herein referred to as SLW mice were maintained under standard conditions. An inbred SLW strain was created by a single backcross to the ddY mouse (Japan SLC, Inc., Shizuoka, Japan), and subsequent mating between heterozygous mice. In this study, the mice used as comparative controls were either heterozygotes or wild type for the non-mutated allele; collectively they are referred to as control. All animal experiments were carried out in accordance with institutional guidelines regarding animal care and handling, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the National Institute of Radiological Sciences.
The TUNEL assay was performed for detection of apoptotic cells using ApopTag® Peroxidase in situ Apoptosis Detection Kit (CHEMICON® International, Inc. CA, USA) according to the manufacturer's instructions.

Statistical analysis
Data are expressed as the means ± SD or SE. The statistical significance of differences in mean values was assessed by Student's t-test.

Results
Developmental delay of spermatogenesis in juvenile but not adult Npr2 mutant mice As previously reported, the Npr2 slw/slw mutant mice exhibited dwarfism and GI distention with gas ( Figure 1A). The testes of Npr2 slw/slw mice were smaller than control testes ( Figure 1B), with the Npr2 slw/slw testis weight reduced by about 50% compared to that of control ( Figure 1C). However, the testis to body weight ratio of Npr2 slw/slw mice was significantly larger in the Npr2 slw/slw than in control mice, reflecting the dwarfism ( Figure 1D).
Histological analyses of testes of Npr2 slw/slw mice revealed no significant differences in overall testis appearance between the control and mutant mice at 0 and 7 dpp (Figure 2A, B, C, D). At 14 dpp, there was evidence of delay in onset and progress of spermatogenesis in Figure 1 Phenotype of testes of Npr2 slw/slw mice. The body size (A) and testis size (B) at 12 dpp of the control litter mate (left) and Npr2 slw/slw mutant (right). Testis weights of the control and Npr2 slw/slw mutants (C), with testes to body weight ratio (D). C and D: n = 4 mice (n = 8 testes) for both control and Npr2 slw/slw mice. The data shown are means (columns) and SDs (bars) (C and D).
Npr2 slw/slw compared to control mice ( Figure 2E, F, and O); additionally vacuolization and presence of eosinophilic cells in the seminiferous epithelium was observed in the Npr2 slw/slw testes, but not in control testes, at 21 dpp ( Figure 2G, H). At 28 dpp, Npr2 slw/slw mouse exhibited seemingly normal spermatogenesis, however the spermatogenic cells were apparently less advanced compared to control. At this age, elongated spermatids were present in control testes, but not observed in the Npr2 slw/slw testes ( Figure 2I and J), indicating delay of spermatogenic progression. However, by 35 dpp, and in adults, the Npr2 slw/slw mice exhibited normal spermatogenesis (O) Delay of spermatogenic onset in Npr2 slw/slw mice. Cumulative bar graph depicting the percent of the most advanced cell types in seminiferous tubules over a whole testis cross-section was compared between control and Npr2 slw/slw testes at 14 dpp. N = 3 different mice of cross sections were observed for examine progression of spermatogenesis. Total number of tubules observed are 779 and 519 for control and Npr2 slw/slw , respectively. The data shown are means (cumulative bar) and SDs (bars). (P and Q) Increased cell death in juvenile testes of Npr2 slw/slw mice. TUNEL assay of Npr2 slw/slw testes at 21 dpp (P). Original magnification x200. Bar indicates 100 μm. Comparison of apoptotic cells in the seminiferous tubules of control and Npr2 slw/slw testes at 21 dpp (Q). N = 4 different mice of cross sections were observed for counting apoptotic cells. Total number of tubules observed are 799 and 502 for control and Npr2 slw/slw , respectively. The data shown are the means (columns) and SEs (bars). and germ-cell composition; spermatozoa were present in the lumens of mutant seminiferous tubules ( Figure 2L and N) as well as those from control mice ( Figure 2K and M).

Apoptosis of Npr2 mutant spermatocytes
A TUNEL assay, performed to detect cell death, revealed that apoptotic cells were abundant in late pachytene spermatocytes in Npr2 slw/slw testes at 21 dpp ( Figure 2P). The number of apoptotic cell in Npr2 slw/slw at 21 dpp was significantly increased compared to that of control ( Figure 2Q).

Paraphimosis in Npr2 mutants
Anatomically, adult Npr2 slw/slw mice exhibited paraphimosis (erectile dysfunction (ED) accompanying a trapped foreskin), while control adult mice did not ( Figure 3A and B). Although the age at which mutant males first exhibited paraphimosis varied among mutant individuals, all adult Npr2 slw/slw mice exhibited paraphimosis. Interestingly, this was a temporary condition; the paraphimosis of Npr2 slw/slw mice did not persist more than one day, although it occasionally recurred. Interestingly, males of another Npr2 mutant strain, Npr2 cn-2J/cn-2J mice, persisted (data not shown).

Discussion
This study provides the first description of the male reproductive phenotype of Npr2-deficient mice. We found that the developmental onset and acquisition of spermatogenic function is delayed in Npr2 slw/slw mutant mice, but that within 5 weeks, there is restoration of apparently normal spermatogenesis. This developmental delay of spermatogenesis suggests that CNP/NPR-B signaling may play a role in initiation of spermatogenic differentiation. However, the mutant mice did not exhibit any apparent mitotic proliferation abnormality as indicated by PCNA staining (data not shown), and as adults, the Npr2 slw/slw mice exhibited normal germ-cell populations. These results are consistent with the previously reported phenotypes of other Npr2 alleles, namely the Npr2-KO [10] and Npr2 pwe mouse models [9]. Thus we conclude that although CNP/ NPR-B signaling is essential for oocyte meiotic arrest in the female mouse [7,17], CNP/NPR-B signaling has different roles in males because spermatogenesis did not suffer meiotic arrest, and thus CNP/NPR-B signaling is not essential for mouse spermatogenesis.
What then might cause the infertility of males bearing homozygous Npr2 mutations? Possibly malnutrition could play a role. Our observations suggest that abnormalities of testis structure (e.g., vacuolization) and delays in onset of spermatogenic function are more prevalent in mice exhibiting the severe GI disorder characteristic of the Npr2 slw/slw mice. In contrast, both Npr2 slw/slw pups with a latent GI phenotype and Npr2 cn-2J/cn-2J mice, which have no apparent GI disorder, showed normal spermatogenic progression (data not shown). Indeed, Npr2 slw/slw mice surviving to adulthood, though rare, exhibited dwarfism and appeared to be healthy, with apparently normal spermatogenesis. Thus we surmise that severe malnutrition from GI abnormalities may cause the spermatogenic delay at 14 dpp and cell death at 21dpp in Npr2 slw/slw mice. Why the Npr2 cn-2J/cn-2J mice do not suffer from these GI problems is not clear, but may depend on the respective genetic backgrounds. These conclusions about the importance of nutrition are consistent with reports on the role of nutrition in growth and development of the reproductive system [16]; moreover, dietary restriction is known to impact negatively on testicular volume, weight and function [18,19].
There may also be other explanations for the infertility of Npr2 mutant males other than physical limitations due to dwarfism. The rare Npr2 slw/slw males that survive to adulthood exhibited unique and abnormal penile phenotypes, including paraphimosis and priapism [10]; this unusual phenotype was also found in adult Npr2 cn-2J/cn-2J and Npr2-KO mice (data not shown). Sildenafil, commonly known as Viagra® (Pfizer), a specific PDE5 inhibitor that increases concentration of cGMP by nitric oxide (NO) via soluble guanylyl cyclase (sGC) [20], has been used as a medical treatment for ED. Because CNP/NPR-B increases cGMP as well as NO/sGC signaling, the phenotypes of Npr2-deficient mice suggest that increase and/or maintenance of cGMP is required for normal erectile function. Our previous report showed that CNP/NPR-B signaling has a critical role in smooth muscle (SM) motility in the gastrointestinal tract [6,14], and therefore prolonged erection in Npr2 slw/slw males is possibly due to abnormal function of the SM of the trabeculae corporum cavernosorum and inability to retain blood flow by contraction due to loss of cGMP production.

Conclusions
In summary, the present study suggests that NPR-Bmediated CNP signaling may play role in male reproductive function, but CNP/NPR-B signaling is not crucial for spermatogenesis. The sub-fertility in adult Npr2 slw/slw males may instead be caused by penile SM dysfunction. Future studies on CNP will provide information to understand the roles of NPR-B-mediated CNP signaling in spermatogenesis and contribute to the development of novel therapeutics for treatment of male infertility syndromes involving ED.