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Gene Locus Leads to Fertility Defects in Male Mice and Men
Department of Pharmacology (K.A.B., M.N.P., M.A.N., G.S.M.), University of Washington School of Medicine, Seattle, Washington 98195-7750; and Greenberg Division of Cardiology, Department of Medicine (D.A.M., D.W., C.T.B.), and Department of Reproductive Medicine and Urology (M.G.), Weill Medical College of Cornell University, New York, New York 10021
Address all correspondence and requests for reprints to: G. S. McKnight, Department of Pharmacology, University of Washington School of Medicine, Box 357750, Seattle, Washington 98195-7750. E-mail: mcknight{at}u.washington.edu.
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ABSTRACT |
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regulatory subunit (RI), PRKAR1. We report here that male mice heterozygous for the Prkar1a gene have severely reduced fertility. Sperm from Prkar1a heterozygous mice are morphologically abnormal and reduced in number. Genetic rescue experiments reveal that this phenotype results from elevated PKA catalytic activity in germ cells as early as the pachytene stage of spermatogenesis. Consistent with this defect in the male mutant mice, sperm from CNC patients heterozygous for PRKAR1A mutations were also found to be morphologically aberrant and decreased in number. We conclude that unregulated PKA activity in male meiotic or postmeiotic germ cells leads to structural defects in mature sperm and results in reduced fertility in mice and humans, contributing to the strikingly reduced transmission of PRKAR1A inactivating mutations by male patients with CNC. |
INTRODUCTION |
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regulatory subunit (RI) of cAMP-dependent protein kinase [protein kinase A (PKA)] and inhibits PKA activity in the absence of cAMP. To explain the multiple neoplasia in CNC patients, it was postulated that PRKAR1A is a tumor suppressor gene; a mutation in one allele and the subsequent loss of heterozygosity of the normal allele results in unregulated PKA activity and tumor formation (4). However, other studies report that haploinsufficiency of the PRKAR1A gene without loss of heterozygosity is associated with cardiac myxomas (3), primary pigmented nodular adrenocortical disease (5), and thyroid tumors (6). Reduced levels of RI can lead to unregulated C subunit basal activity (7) and may also shift the predominant holoenzyme from one containing RI to one containing the type II regulatory subunit (RII) (3). Because RII-containing PKA holoenzymes bind more tightly to many A kinase-anchoring proteins (8, 9), a shift in the subcellular localization of PKA could be another factor in the changes in cell cycle control and tumor formation in CNC patients. Our studies with animals containing a null mutation in one allele of Prkar1a suggest that unregulated PKA activity and not relocalization of subcellular PKA contributes to the fertility defects that are observed in the males. Transmission of CNC occurs more frequently through the female parent, but the mechanism for this is unknown (10, 11). Our results demonstrate that male mice with a null mutation in one allele of Prkar1a are subfertile as a result of abnormal sperm morphology and reduced sperm count. Semen analyses from seven male CNC patients with mutations in PRKAR1A also show a sperm morphology phenotype similar to the mutant mice. These data suggest a common mechanism underlying male infertility in mice and men with haploinsufficiency of PRKAR1A.
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RESULTS |
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+/– Male Mice) gene in mice is embryonic lethal in the homozygous state. As described in a previous report, the RI–/– embryos fail to develop a functional heart tube and are resorbed at embryonic d 10.5. This defect in development is largely due to unregulated C subunit activity because it could be rescued by a genetic depletion of C subunit in R–/–embryos (7). Mating of RI heterozygotes, to generate RI–/– embryos, revealed that RI+/–males, but not females, had reduced fertility when back-crossed to a C57BL/6 background. A fertility study in which RI+/– males on a high C57BL/6 background (94% or 99%) were mated with wild-type C57BL/6 females confirmed that these RI+/– males are severely subfertile (Table 1
). Mature sperm from the cauda epididymides of these males were fragile with broken heads and ruptured tails (Fig. 1), were reduced in number, were less motile, and were defective in fertilizing zona pellucida (zp)-intact and zp-free eggs (Table 1). No significant differences were observed in testes or seminal vesicle weight or serum content of LH or testosterone. FSH was decreased slightly (33%) but significantly.
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+/– males that were on a 50:50 background showed no deficits in their fertility as each plugged female produced a normal litter size (7 ± 1 pups, mean± SEM). Although most severe on a C57BL/6 background, the fragility of the sperm was clearly evident on a mixed (50:50) 129Sv/J:C57BL/6 background and on mice backcrossed into the 129Sv/J background (Fig. 1E
). The normal fertility of the RI+/– males on the 50:50 background suggests that although approximately half of the sperm are headless, a sufficient percentage of sperm are morphologically normal and capable of fertilizing eggs. These data indicate that RI+/– males on a high C57BL/6 background are subfertile due to sperm defects and reduced sperm number and motility. In addition, genetic modifier(s) contribute to the severity of the phenotype, rendering most RI+/– males on a C57BL/6 background completely infertile.
We examined testes and epididymides of RI+/– mice to determine the basis for the sperm abnormalities. RI+/– and wild-type testes sections were similar throughout most stages of spermatogenesis and spermiogenesis. However, in testis sections of RI+/– as young as 8 wk of age, stage I round spermatids contained large clear areas in the nucleus devoid of chromatin (Fig. 2, A–D). A similar nuclear phenotype was observed in stage V–VI round spermatids in casein kinase 2 catalytic subunit (Csnk2a2) –/– mice (12) and, combined with nuclear envelope abnormalities and reduced epididymal sperm count, it was suggested that Ck2' deficiency impairs the phosphorylation of nuclear proteins resulting in cell death. Similarly, the reduced sperm count and nuclear abnormalities in RI+/– animals may be a result of altered phosphorylation levels of nuclear proteins by PKA.
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+/– contained premature chromatin focal condensations not found in wild-type spermatids (Fig. 2, E and F). Precocious nuclear chromatin condensation can be triggered by premature synthesis of the nucleoprotein protamine 1 (13), leading to sterility in mice, and it has been reported that infertility in a subset of human patients correlates with abnormal protamine content (14). We have found no change in protamine gene expression by array analysis (data not shown) and no change in protein expression in RI+/– mice. However, we cannot exclude a more subtle temporal change in protamine expression that could result in the premature chromatin condensation observed in stage IX spermatids.
A third difference appeared in approximately 60% of older (at least 19 wk old) RI+/– animals: the seminiferous tubules of the testis were distended (Fig. 2, G–J) with germ cell sloughing in some of the tubules (Fig. 2J). In addition, the corpus epididymis was enlarged (Fig. 3, A and B), and spermatic granulomas were present (Fig. 3, C and D). A similar phenotype is induced experimentally in laboratory animals by vasectomy or by injection of spermatozoa into connective tissues (15, 16). Because no obstructions of the vas deferens were observed in RI+/– animals, we suggest that defects in the epididymis may leak spermatozoa from the ductal lumen, induce inflammatory cell responses, and evoke formation of a spermatic granuloma and back pressure-induced testicular atrophy. Although some CNC patients have large-cell calcifying Sertoli cell tumors, no evidence of large-cell calcifying Sertoli cell tumors has been observed in testes from RI+/– mice.
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+/– Fertility Defects haploinsufficiency causes fertility defects in male mice by a decrease in the content of RI protein and a subsequent increase in constitutive (basal) PKA activity in germ cells. The R2C2 tetrameric holoenzyme is in the inactive state until cAMP levels are elevated and the active C subunit is released. The loss of one allele of the RI gene lowers RI mRNA and RI protein synthesis and, as a consequence, some C subunit might be left in the unbound and active state. Because RI+/– is the major PKA-regulatory subunit expressed in premeiotic and meiotic male germ cells (17, 18), this stage of spermatogenesis is the likely time when kinase activity is unregulated in RI+/–. If elevated PKA results in defective sperm in RI+/– animals, then genetically reducing the amount of kinase activity might be expected to partially rescue the phenotype. We crossed RI+/– animals with C2+/– mice engineered to have reduced PKA activity only in meiotic and postmeiotic germ cells. The C2 isoform is an alternatively spliced product of the Prkaca gene (19, 20) and is expressed exclusively in male germ cells. C2 mRNA appears first at the midpachytene stage of spermatogenesis and is the only catalytic subunit found in mature sperm (19, 20, 21). C2 homozygous null males are infertile due to a complete loss of PKA activity in sperm although sperm morphology is normal. However, C2 heterozygous males are fertile with no apparent defects in sperm number, motility, or morphology (21).
For the attempted rescue, RI+/–;C2+/– adult males (97% C57BL/6) were generated by crossing RI+/– females with C2+/– males. Sperm from these double heterozygote mice on a high C57BL/6 background were morphologically indistinguishable from wild type (Fig. 4, A and B), demonstrating a rescue of the RI+/– mutant fragile sperm phenotype. Moreover, the sperm were capable of fertilizing eggs and generating offspring as efficiently as wild-type sperm (data not shown). However, spermatic granulomas in the epididymis and testicular atrophy were still observed in approximately 60% of double heterozygote males, which is similar to their incidence in RI+/– animals. Because C2 is not expressed in the epididymis, the genetic rescue of the spermatic granulomas phenotype was not expected or observed.
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+/–;C2+/– animals (Fig. 4, C and D). This reduction in PKA activity correlates with the restoration of normal sperm morphology and fertility and supports the hypothesis that elevated PKA activity exclusively in a subset of germ cells as early as the midpachytene stage of spermatogenesis causes sperm and fertility defects. However, we were unable to detect biochemically an increase in basal PKA activity in either adult sperm or testes from RI+/– animals (Fig. 4, C and D). The adult testis contains germ cells at all stages of development, and we postulated that basal PKA activity is elevated in RI+/– in a subpopulation of developing germ cells in which RI is the primary regulatory of PKA activity. Therefore, we measured PKA activity in testis from juvenile animals (postnatal d 10 and postnatal d 25) before the appearance of RII in elongated spermatids. These assays were also unable to detect an increase in basal PKA activity in juvenile RI+/– testis (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).
Compensation by Other Regulatory Subunits in RI+/–
Compensation by the other regulatory subunits may regulate PKA activity when RI levels are reduced and contribute to the infertility phenotype. In developing germ cells, RI and RII are the primary regulatory subunits expressed (17). RII mRNA is normally induced in elongating spermatids (18) but could be prematurely induced to regulate PKA activity in RI+/– developing germ cells. In RI+/– testes, but not in mature sperm, RI protein levels were reduced and RII protein levels were elevated (Fig. 5, A and B), suggesting that RII may compensate and partially regulate basal kinase activity in RI+/– immature germ cells. However, Northern analysis indicated that the shorter (2.4 kb) testis-specific RII mRNA is not prematurely induced in RI+/– testis but is expressed at a time point coincident with the appearance of elongating spermatids and is similar to wild-type expression (Fig. 5C). The larger (6.0 kb) RII transcript was evenly expressed across all time points and did not differ between wild-type and RI+/–. Because RII mRNA levels are unchanged but protein levels are elevated in RI+/– testes, it is likely that posttranscriptional stabilization of RII protein may be involved and the increased RII protein levels may serve to help regulate PKA activity when RI levels are reduced.
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compensation contributes in any way to the abnormal morphology of RI+/– sperm and the infertility phenotype. We had previously established a line of mice with a null mutation in the RII gene, and these animals were shown to have normal sperm morphology and motility and fertility (22). RI+/– animals were crossed with RII-deficient mice, and RI+/–/RII–/– males were generated. The RI+/– males on the RII null background exhibited the same sperm abnormalities (Fig. 6, A and B) and infertility as RI+/– animals. RI+/–/RII–/– males (n = 2) each plugged four wild-type females but generated one pup total compared with 74 pups from similarly mated wild-type males (n = 2). These data indicate that the subfertility and abnormal sperm morphology of RI+/– animals are not dependent on the presence of RII and rule out a compensation mechanism as a cause of the subfertility phenotype. This finding, coupled with the genetic rescue experiment with C2+/– animals, suggests that reduced levels of RI protein in the RI+/– animals result in inappropriate C subunit activity and sperm fragility.
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+/– mouse, results from abnormal sperm morphology and reduced sperm count. Ejaculates from CNC patients contained a high percentage of abnormal sperm characterized by head or tail defects. The presence of immature germ cells was also observed (Fig. 7). Almost half (three of seven) of the patients were azoo- or oligospermic. The sperm defects were observed in four unrelated probands and in all three CNC patients analyzed in family YZZ. These observations indicate that male CNC patients with haploinsufficiency of the PRKAR1A gene have reduced fertility and consequently reduced transmission of the mutation as a result of sperm morphology defects and azoo- or oligospermia. CNC patients without PRKAR1A mutations were not available for study. However, no evidence of male infertility was observed in a large CNC family carrying a mutation in the perinatal myosin heavy-chain gene (MYH8) (25).
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+/– mice, the sperm morphology changes were not identical. The majority of sperm from RI+/– mice were too fragile to isolate intact, whereas most sperm from CNC patients were intact. These differences might be species specific or a result of the differences in isolation for mouse sperm (cauda epididymal dissection) vs. the human sperm (ejaculate). |
DISCUSSION |
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+/– mutations. Genetic rescue experiments demonstrate that elevated PKA activity, and not relocalization of the kinase in germ cells, produces the sperm defects in mice. This increase in PKA activity interferes with a developmental pathway required for the integrity of the sperm head and tail. In male germ cells, PKA affects gene expression (26), nucleosomal protein activity (27, 28), and sperm motility (29). The nuclear changes evident in the stage I round spermatids (chromatin condensation at the margins of the nuclei) and premature focal chromatin condensation (stage IX) suggest that PKA is altering nuclear function.
The genetic rescue experiment supports the hypothesis that elevated PKA activity exclusively in germ cells as early as the midpachytene stage of spermatogenesis, when C2 is expressed, causes fragile sperm. The inability of the kinase assay to detect a change in PKA activity in adult or juvenile RI+/– testis is consistent with the finding that free C subunit is unstable and rapidly degraded when not tightly complexed with R subunit (30, 31). This is supported by previous results from our laboratory in which a mutation was made in the C subunit to lower its binding affinity for the R subunit, resulting in constitutive C subunit activity (32). When this constitutively active C subunit was expressed in the liver, no change in basal PKA activity was observed in liver extracts even though a phenotype predicted by elevated PKA activity (altered glucose homeostasis, glycogen storage, and fructose 2,6-bisphosphate levels) was evident.
The inability of C2+/– mice to rescue the granulomas and testicular atrophy of the RI+/– mouse suggests that the RI+/– epididymis phenotype reflects a somatic cell defect. Whether this is due to an alteration of PKA activity or its subcellular localization is not known. Alterations in fluid absorption and/or secretion in the efferent duct, a phenotype of ER–/– (33) males, may contribute to pressure changes that lead to spermatic granuloma formation and back-pressure atrophy of the testis. PKA inhibits the sodium/hydrogen exchanger 3 (NHE3) in epithelial cells of the proximal kidney (34). NHE3 is also expressed in the efferent duct and epididymis where it is involved in the absorption of tubular fluid (35). One possibility is that unregulated PKA in nonciliated cells of the efferent duct in RI+/– mice phosphorylates and inhibits the activity of NHE3. Decreased absorption could then dilate epididymal tubules and initiate back-pressure atrophy of the testis.
Infertility affects about 5% of the male population and yet, in most cases, the molecular cause remains uncertain. We have shown that haploinsufficiency of the RI gene in both mice (Prkar1a) and humans (PRKAR1A) is deleterious to the structural integrity of mature sperm and, in mice, dramatically reduces the ability of the sperm to fertilize eggs. The genetic rescue experiments support the hypothesis that unregulated PKA activity in germ cells leads to aberrations in sperm structure and the resulting infertility. These results reveal the toxicity of unregulated kinase activity in developing male germ cells and are in sharp contrast to the phenotype of C2-deficient male germ cells, which undergo normal spermatogenesis and spermiogenesis yet are immotile (21). These findings demonstrate the significance of the PKA signaling pathway in male fertility and support previous work showing that components of this pathway, such as adenylyl cyclases (36, 37) and A kinase-anchoring proteins (38), are likely candidates affecting male fertility in humans.
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MATERIALS AND METHODS |
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+/– mice were derived as previously described (7). Animals were 94% and 99% C57BL/6 with the remaining background as 129Sv/J, with the exception that for the sperm morphology analysis on different C57BL/6 backgrounds, 50% and 6% C57BL/6 animals were additionally used. In all studies, wild-type (control) animals and RI+/– animals are on the identical genetic background, either 94% or 99% C57BL/6. Female 6- to 8-wk-old C57BL/6 mice were purchased from Jackson ImmunoResearch Laboratories, Inc. (Bar Harbor, ME) for fertility studies, and 3-wk-old B6C3 mice were purchased from Taconic Farms, Inc. (Germantown, NY) for in vitro fertilization studies. All experiments were carried out in accordance with guidelines established by the Institutional Animal Care and Use Committee at the University of Washington.
Mouse Fertility Assessment, Sperm Swimout, and Motility Assay
A fertility study and sperm motility analysis were performed as described previously (22). To assess sperm motility, mature spermatozoa were obtained from the cauda epididymis. The analysis was performed on video-taped sperm samples obtained from 10- to 20-wk-old mice using an automated Hamilton-Thorn Motility Analyzer (Hamilton-Thorn Research Inc., Danvers, MA). A slide chamber of 50 µm depth was used, and motile sperm were tracked at 60 frames/sec. The observer was unaware of the genotype of the sperm. Twenty fields were recorded for 5 sec each, and percent motility was evaluated on a minimum of 100 sperm from the recorded sperm samples.
Sperm Morphology and Count
Sperm from the cauda epididymis were released into capacitating media as described above. A sperm suspension was placed on a glass slide (Superfrost Plus, VWR, West Chester, PA) by gently smearing 10 µl of the suspension across the top of the slide using the edge of a second glass slide. The slides were air dried. Sperm morphology was observed after histological staining or propidium iodide staining. For histological evaluation, sperm were stained (Diff-Quik Stain Set; Dade Behring, Inc., Newark, DE) and viewed at x40 under a Nikon light microscope (Nikon, Inc., Melville, NY). One hundred sperm were counted per animal. Sperm head morphology was evaluated after propidium iodide staining (20 µg/ml) and DNA-containing heads were visualized with a Nikon fluorescent microscope at x40. All heads stained with propidium iodide were normal in shape whether they were attached to a flagellum or not. Sperm count was estimated by counting the total number of flagellum removed from both cauda epididymides on a glass slide prepared as described for the histological evaluation.
Electron Microscopy
Sperm were isolated from the cauda epididymis as described above. Harvested sperm were pelleted at 600 x g, and the supernatant was removed. The sperm were fixed in 3% gluteraldehyde in 0.1 M PBS for 24 h at 4 C. Sperm were collected on Nucleopore filters or poly-L-lysine-coated glass cover slips, dehydrated in a graded ethanol series, subjected to critical point drying, and coated with gold/palladium. Samples were examined with a JEOL JSM 6300F scanning electron microscope at an accelerating voltage of 15 kV at the Electron Microscopy Laboratory at the University of Washington.
Western Blot and Kinase Assay
Sperm were obtained from the cauda epididymides in PBS and diluted in sample buffer. Western blot analysis and kinase assay were performed on these samples as previously described (22, 39).
RNA Isolation and Analysis
Testes were removed from animals at postnatal days 24, 27, 30, and 35 and RNA was isolated by using the RNeasy Kit (QIAGEN, Chatsworth, CA) according to the manufacturer’s protocol. The samples were denatured in 20 mM MOPS, pH 7.0, 1 mM EDTA, 5 mM sodium acetate, 2.2 M formaldehyde, and 50% formamide at 65 C for 15 min. The samples (5 µg total RNA) were loaded on a 1.2% agarose gel and run in the same buffer without formamide. The gel was then blotted to nitrocellulose and hybridized overnight with a nick-translated mouse cDNA probe (0.8 kb HindIII-SmaI fragment: all within the open reading frame for mouse RII). Equal loading was visualized and confirmed by staining the 28S and 18S ribosomal bands with 0.4% methylene blue.
In Vitro Fertilization
B6C3 females (3 wk old) were ip injected with pregnant mare serum (2.5 IU) followed 48 h later with human chorionic gonadotropin (2.5 IU). The females were euthanized 13 h after the second injection, and their oviducts were removed. The oocyte-cumulus complexes were isolated, and the cumulus was removed in hyaluronidase (10 mg/ml) in M2 medium (Sigma Chemical Co., St. Louis, MO). A proportion of the eggs were subsequently treated with acidic Tyrode’s solution (Sigma) to remove the zp. Sperm were obtained as described above in human tubal fluid media (Irvine Scientific, Irvine, CA) supplemented with 0.5% BSA. Sperm (10 µl) at 1 x 106 sperm/ml were added to a 250 µl drop of human tubal fluid containing five cumulus-free eggs and covered with light mineral oil (Sigma, embryo tested). Dishes were placed in an incubator with 5% C02/95% air at 37 C overnight. The next morning, the number of two-cell embryos was scored.
Serum Hormone Measurements
Blood was collected from euthanized animals by cardiac puncture, and serum was analyzed by RIA for mouse LH, mouse FSH, and testosterone by the University of Virginia Center for Reproduction Ligand Assay and Analysis Core Facility.
Semen Analysis from CNC Patients
After providing informed consent per institutional guidelines, men with known PRKAR1A mutations provided semen specimens for analysis within 2 h of collection by the Cornell University Medical College Infertility Laboratory or the In Vitro Fertilization Unit at the North Karelian Central Hospital in Joensuu, Finland. Manual light microscopic evaluation of sperm concentration, motility, and morphology was performed. Analysis was performed by technicians unaware of the patient’s genotype or medical history.
Statistical Analysis
Unpaired t tests were performed when comparing between wild-type and mutant groups
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The authors have nothing to declare.
First Published Online May 25, 2006
Abbreviations: C, Catalytic subunit; CNC, Carney complex; NHE3, sodium/hydrogen exchanger 3; PKA, protein kinase A; PRKAR1, gene encoding the PKA RI; RI, regulatory subunit I; RII, type II regulatory subunit; zp, zona pelucida.
Received for publication February 2, 2006. Accepted for publication May 17, 2006.
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REFERENCES |
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of protein kinase A (PRKAR1A): a tumor-suppressor gene for sporadic thyroid cancer. Genes Chromosomes Cancer 35:182–192[CrossRef][Medline]’: failure of germ cell survival and characteristic modifications of the spermatid nucleus. Mol Reprod Dev 66:190–201[CrossRef][Medline] mRNA expressed specifically in spermatogenic cells. Mol Biol Cell 11:3031–30442 orchestrates cAMP signaling for male fertility. Proc Natl Acad Sci USA 101:13483–13488 regulatory subunit in patients with the Carney complex. Nat Genet 26:89–92[CrossRef][Medline]This article has been cited by other articles:
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