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Published-Ahead-of-Print January 24, 2007, DOI:10.2164/jandrol.106.002006
Journal of Andrology, Vol. 28, No. 3, May/June 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.106.002006

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Abnormal Morphology of Spermatozoa in Cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase (CYP17) Deficient Mice

YING LIU*, LUIS E. DETTIN*, JANET FOLMER{dagger}, BARRY R. ZIRKIN{dagger} AND VASSILIOS PAPADOPOULOS*

From the * Department of Biochemistry & Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC; and the {dagger} Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland.

Correspondence to: Dr Vassilios Papadopoulos, Georgetown University Medical Center, Department of Biochemistry & Molecular and Cellular Biology, 3900 Reservoir Road NW, Washington DC, 20057 (e-mail: papadopv{at}georgetown.edu).
Received for publication October 24, 2006; accepted for publication January 17, 2007.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase (CYP17) is crucial for cortisol and sex steroid biosynthesis. In a previous study we examined CYP17 function by generating mice with a targeted CYP17 deletion. We found that in addition to its role in steroid biosynthesis, CYP17 is present in germ cells. In the present study we examined the effect of CYP17 on sperm morphology. Disorganization of the sperm midpiece, small sperm mitochondria with reduced inner membranes and matrix, and irregular sperm shape were found to be associated with the CYP17 gene deletion. Treating the mice carrying the CYP17 deletion with testosterone did not alleviate the observed sperm phenotypes, suggesting that CYP17 acts in a testosterone-independent manner. These results suggest that CYP17, in addition to its role in androgen formation, is critical for proper mitochondrial architecture and sperm morphology and thus for sperm function and normal fertility.

     Key words: Gene deletion, steroidogenesis, mitochondria architecture, fertility


Cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase (CYP17; EC 1.14.99.9) is a bifunctional microsomal monooxygenase that mediates the 17{alpha}-hydroxylation of pregnenolone or progesterone to yield 17{alpha}-OH pregnenolone or 17{alpha}-OH progesterone, respectively. Cleavage of the C17, 20 bond leads to cortisol and sex steroid biosynthesis (Hall, 1986; Mellon and Griffin, 2002; Miller, 2002). Human CYP17 deficiency results in impaired cortisol, androgen, and estrogen production, as well as mineralocorticoid overproduction, which potentially leads to hypertension, pseudohermaphroditism, and delay in sexual maturation (Kater and Biglieri, 1994; Yanase, 1995; Rainey et al, 2002). CYP17 is also a potent oxidant with catalytic properties distinct from its 17{alpha}-hydroxylase/17, 20-lyase activity (Lieberman and Warne, 2001). Our in vitro studies demonstrated that CYP17 has squalene monooxygenase (epoxidase) activity, critical for cholesterol biosynthesis (Liu et al, 2005b).

Recently we investigated the function of CYP17 in vivo by generating mice with a targeted deletion of CYP17. An 80% reduction in intratesticular and circulating testosterone levels occurred in chimeric mice carrying the CYP17 deletion (Liu et al, 2005a). Interestingly, the testosterone that remained was sufficient to support spermatogenesis. However, the male chimeras consistently failed to generate heterozygous CYP17 mice. Unexpectedly, CYP17 was found to be present in germ cells. Moreover, more than 50% of the chimeric mice sperm were morphologically abnormal, and many had impaired motility (Liu et al, 2005a).

The objective of the present study was to further determine the effect of the CYP17 deletion on sperm morphology and fertility. Using hormone replacement, we also addressed the question of whether changes in the sperm might be the consequence of reduced intratesticular androgen. We show that CYP17 is necessary for the structure and organization of the sperm mitochondria and that the mitochondrial defects observed in CYP17 chimeric mice were not alleviated by androgen treatment.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and Treatments

Mice with the CYP17 gene deletion were generated and characterized as previously described (Liu et al, 2005a). Adult mice were used for the morphologic studies described herein. To examine the effect of testosterone replacement on sperm function and fertility in the chimeric mice, newborn chimeric and wild-type (control) male pups (6–7 days old) were injected subcutaneously with testosterone (10 µg testosterone/50 µL/5 g body weight) or vehicle (peanut oil) every 2 days until the mice reached adulthood (2 months), as described previously (Chapman, 2001; James and Nyby, 2002; Martinez-Sanchis et al, 2002). Testosterone and peanut oil were from Sigma (St Louis, MO). All mice were mated with wild-type (C57BL/6 strain) females to generate offspring. All animal studies were approved by the Georgetown University Animal Care and Use Committee.


Figure 1
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  		Figure 1. (A) Hematoxylin and eosin–stained paraffin sections of wild-type (40x) and (B) cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase (CYP17) chimeric mice (40x). There are no noted differences between wild-type and CYP17 chimeric mice. (C) Leydig cells from wild-type (100x) and (D) chimeric mice (100x), in plastic-embedded testes, also do not have any observable differences.

 
Hematoxylin and Eosin Staining

Testes were fixed in 4% paraformaldehyde. Paraffin-embedded testes from wild-type (C57BL/6 strain) and CYP17-deleted chimeric adult mice (Liu et al, 2005a) were prepared as described in Thuillier et al (2003). Sections were stained with hematoxylin (Sigma) and eosin (Fisher Scientific, Fairlawn, NJ) (HE).

Leydig Cell Morphology

For ultrastructural analyses, mice were perfused through the heart with 5% glutaraldehyde in 0.1 M sodium cacodylate buffer. After initial fixation, testis blocks were immersed in 5% glutaraldehyde overnight, postfixed in cacodylate-buffered 1% osmium tetroxide, washed, dehydrated, and embedded in Epon 812. Semithin sections (1 µm) were mounted on glass slides and stained with toluidine blue. Brightfield images were captured with a Nikon Eclipse 800 microscope system equipped with a Princeton Instruments CCD camera (Trenton, NJ) and digitized with IPLab software.

Sperm Morphology and Ultrastructure

For light microscopy analysis of epididymal spermatozoa, epididymes from wild-type (n = 4) and chimeric (n = 5) mice carrying the CYP17 gene deletion were dissected. Sperm from cauda epididymes were extruded into phosphate-buffered saline and observed under phase-contrast microscopy.


Figure 2
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  		Figure 2. Light microscopy analysis of sperm extruded from the epididymides of (A–C) wild-type and (D–K) cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase chimeric mice. In comparison with spermatozoa from wild-type mice, spermatozoa from chimeric mice were characterized by abnormal morphology, including sperm bent over in the midpiece (D, G); head and neck attached to the midpiece (J, K); loops formed (E), and/or head touching the midpiece (D, E, F, H, I). A, D, E: 20x magnification; B, C, F–K: 40x magnification. Data shown are representative of 4 wild-type and 5 chimeric mice.

 
For ultrastructural studies, wild-type (C57BL/6 strain) and high-percentage chimeric adult male mice (n = 5) carrying the CYP17 gene deletion (Liu et al, 2005a) were sacrificed. Cauda epididymes were excised and fixed with 5% glutaraldehyde in collidine buffer (0.05 M, pH 7.4). After 30 minutes the samples were cut into 1-mm cubes and placed in fixation solution for an additional hour before being washed 3 times with collidine buffer, and then blocks were postfixed in a 1:1 mixture of 4% osmium tetroxide and 2% potassium ferrocyanide for 2 hours. Samples were embedded in Spurr embedding medium (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were stained with uranyl acetate and lead citrate. The sections were examined and photographed with a Digital Hitachi H-7600 transmission electron microscope (Hitachi, Tokyo, Japan).


Figure 3
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  		Figure 3. Electron micrographs of longitudinal sections of sperm obtained from cauda epididymes of (A–F) wild-type and (G–M) cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase (CYP17) chimeric mice. The sperm from wild-type mice show the midpiece mitochondrial sheath (A: 7000x; B–E: 10 000x; D: 6000x) and the junction of the middle and principal pieces with the annulus (F, arrows: 8000x). Sperm from CYP17 chimeric mice (M; 10 000x) show the normal junction of the middle and principal pieces, which show the normal annulus structure (thick arrow). However, the mitochondria in the middle piece mitochondrial sheath show abnormal morphology (arrows): L (10 000x), K (10 000x), H (12 000x), irregular shape; J (10 000x), very small size; I and L (10 000x), shrunk; G (3500x), shows bent sperm with irregular mitochondria in the middle piece. Data shown are representative of 4 wild-type and 5 chimeric mice.

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Observation of HE-stained, paraffin-embedded testis sections revealed no apparent differences in spermatogenesis between high-percentage (75%–80%) chimeric and wild-type mice (Figure 1A and B). There also were no observable differences between the Leydig cells seen in wild-type and chimeric mice (Figure 1C and D). Approximately 90% of the sperm collected from the epididymides of wild-type mice appeared normal by light microscopic examination (Figure 2A through C). However, a number of abnormalities were seen in the sperm of chimeric mice (Figure 2D through K), including: 1) sperm head touching the flagella, 2) sperm bent over, and/or 3) loops in the sperm midpiece area.

Electron microscopy (EM) was used to examine sperm ultrastructure in wild-type and chimeric mouse sperm. The most apparent differences were seen in the sperm midpiece. Figures 3 and 4 show representative EM longitudinal sections of spermatozoa from wild-type animals (Figures 3A through F and 4A through C), displaying normal mitochondrial sheathes in the middle piece and unaffected annuli (ring-like structures) at the junction of the middle and principal pieces. No differences were seen in the sperm annuli between chimeric and wild-type mice (Figures 3G through M and 4D through G), and no differences were seen in the principal pieces. However, differences were seen in the middle pieces. In sperm from chimeric mice, the middle pieces were irregularly shaped, small, and disorganized. Cross-sections of chimeric CYP17 and wild-type sperm usually showed abnormal mitochondrial shape and organization in the middle pieces of chimeric mouse sperm (Figure 5D through G). These results were consistent with our earlier report that the majority of sperm of chimeric mice appeared morphologically abnormal by light microscopy and that many had impaired motility (Liu et al, 2005a). No other differences were seen even at high magnification (40 000x–60 000x).


Figure 4
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  		Figure 4. Electron micrographs of longitudinal sections of sperm obtained from epididymes of (A–C) wild-type and (D–G) cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase chimeric mice. The junction between middle and principal pieces with annulus are shown in A (7000x) and D (5000x); kidney-shapes mitochondria (arrows) can be seen in D (10 000x), E (10 000x), F (8000x), and G (8000x). Data shown are representative of 4 wild-type and 5 chimeric mice.

 

Figure 5
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  		Figure 5. Electron micrographs of cross-sections of sperm obtained from epididymes of (A–C) wild-type and (D–G) cytochrome P450 17{alpha}-hydroxylase/17, 20-lyase (CYP17) chimeric mice. A (6000x), cross sections at the level of the middle (*) and principal (**) pieces. B (10 000x) and C (30 000x) are at higher magnification and show the normal structural details in the middle and principal pieces; sperm from CYP17 chimeric mice showed normal structure in the principal but abnormal in the middle piece (D, 3500x). E (10 000x), F (10 000x), and G (8000x) show irregularly organized and missing mitochondria in the middle.

 

To examine the possibility that CYP17-induced sperm morphology changes were due to decreased androgen levels, newborn chimeric male pups were treated with testosterone for 2 months. Testosterone treatment did not rescue the sperm defects (Figure 6) or alter fertility status (data not shown). These observations suggest that CYP17 may affect sperm development directly rather than exerting its effects indirectly through reduced androgen levels.


Figure 6
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  		Figure 6. Testosterone treatment of chimeric male mice with the CYP17 gene deletion. Newborn chimeric male pups (6–7 days old) were injected subcutaneously with either (A) vehicle or (B) testosterone, and sperm was collected and viewed by light microscopy (40x). Data shown are representative of control (n = 2) and testosterone-treated (n = 3) chimeric mice.

 


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We recently demonstrated that chimeric mice carrying the CYP17 gene deletion have reduced testosterone concentrations both in blood serum and in the testis (Liu et al, 2005a). As evidenced by the generation of wild-type black C57BL/6 mice, the testosterone remaining in the testes of the chimeric animals was sufficient to support spermatogenesis (Liu et al, 2005a). This was not surprising because at least qualitatively complete spermatogenesis can occur at intratesticular testosterone concentrations as low as 2% of the control level (Zhang et al, 2003). However, the male mouse chimeras consistently failed to generate heterozygous CYP17 mice (Liu et al, 2005a). Unexpectedly, CYP17 was found to be present in the germ cells of the mice (Liu et al, 2005a), as also has been reported for the Japanese black bear (Okano et al, 2003).

Sperm morphology has been shown to correlate with sperm function in fertilization and thus to have prognostic value in assisted reproduction. For example, abnormal mitochondrial architecture in mature sperm has been correlated with loss of motility and fertilization potential (Chemes and Raue, 2003). During spermatogenesis mitochondria undergo a dramatic reorganization in the middle piece of the sperm tail that results in a helical alignment of similarly shaped and sized organelles along the sperm axoneme, referred to as the mitochondrial sheath (Kissel et al, 2005). Sperm dysfunction has been associated with the absence of mitochondria around the axoneme, with the middle piece appearing thin and bent and with an abnormal fibrous sheath extending up to the neck region, thus preventing mitochondrial assembly around the axoneme (Zamboni, 1992). Ultrastructural analyses of abnormally shaped spermatozoa often show structural irregularities in the mitochondrial sheath (Olson et al, 2004). Infertile human males also are reported to exhibit ultrastructural defects in the mitochondria of their middle pieces (Foresta et al, 2002).

Such previous studies provided the rationale for our analysis of sperm ultrastructure in the CYP17 knockout mice. Our intent was to understand how CYP17 affects sperm morphology and motility. To this end, EM was used to analyze spermatozoa obtained from CYP17 chimeric and wild-type mice. The CYP17 deletion was found to have a profound effect on mitochondrial architecture. In particular, structural irregularities and mitochondrial sheath disorganization in the midpiece were seen in sperm from the chimeric in comparison with the wild-type mice. In particular, the mitochondria of sperm from the chimeric mice were small, abnormally shaped, and disorganized. Testosterone supplementation from birth to adulthood did not rescue abnormal sperm morphology, suggesting that in addition to its critical role in androgen formation, CYP17 is necessary for proper sperm development and function. These results suggest that the effects of the CYP17 deletion may be direct rather than through reduced amounts of androgen.

Kissel et al (2005) recently reported abnormalities in the morphology of sperm from Sept4 gene knockout mice, including alteration of mitochondrial architecture and annulus formation, as well as effects on the removal of residual cytoplasm during sperm maturation. Although the CYP17 deletion also resulted in altered sperm mitochondrial architecture, there were no apparent annulus or cytoplasm abnormalities.

In previous studies we reported that CYP17, in addition to its 17{alpha}-hydroxylase/17,20-lyase activity, has squalene monooxygenase (epoxidase) activity that is critical for cholesterol biosynthesis (Liu et al, 2005b). MA-10 Leydig cells have very low amounts of CYP17. Nonetheless, we observed that MA-10 Leydig cells in which CYP17 was knocked out had reduced ATP levels compared with control MA-10 cells (data not shown), suggesting that CYP17 may play a role in mitochondrial function in these cells. Taken together with the results presented herein, these findings suggest that the squalene monooxygenase (epoxidase) activity of CYP17 may affect mitochondrial function, leading to altered sperm morphology and infertility.

In conclusion, the data presented here provide evidence that CYP17 plays a critical role in the organization and structure of the sperm mitochondria. These mitochondrial defects, rather than an altered androgen environment, likely cause the defects in sperm motility and altered fertilization potential in mice with a targeted CYP17 deletion.


   Acknowledgments
 
We would like to thank Dr Zeqiu Han for assisting with animal dissection studies and the Transgenic Shared Resource at the Lombardi Comprehensive Cancer Center (Georgetown University), supported by grant P30 CA51008-13 from the National Cancer Institute.


   Footnotes
 
This work was supported by grant IBN-0110711 from the National Science Foundation.


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