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Reproduction (2007) 133 395-403
DOI: 10.1530/REP-06-0180
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

Deficiency of co-chaperone immunophilin FKBP52 compromises sperm fertilizing capacity

Jiyoung Hong1, Sung Tae Kim1, Susanne Tranguch2, David F Smith4 and Sudhansu K Dey1,2,3

1 Departments of Pediatrics, 2 Cell and Developmental Biology, 3 Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA and 4 Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, Arizona 85259, USA

Correspondence should be addressed to S K Dey, Division of Reproductive and Developmental Biology, Department of Pediatrics, Vanderbilt University Medical Center, MCN-D4100, Nashville, Tennessee 37232-2678, USA; Email: sk.dey{at}vanderbilt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
FKBP52 is a member of the FK506-binding family of immunophilins and serves as a co-chaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Male mice missing Fkbp52 are infertile, and this infertility has been ascribed to compromised sensitivity of the anterior prostate, external genitalia, and other accessory sex organs to androgen. Here, we show additional defects contributing to infertility. We found that epididymal Fkbp52–/– sperm are sparse often with aberrant morphology, and they have reduced fertilizing capacity. This phenotype, initially observed in null males on a C57BL/6/129 background, is also maintained on a CD1 background. Expression studies show that while FKBP52 and androgen receptor are co-expressed in similar cell types in the epididymis, FKBP52 is also present in epididymal sperm flagella. Collectively, our results suggest that reduced number and abnormal morphology contribute to compromised fertilizing capacity of Fkbp52–/– sperm. This study is clinically relevant because unraveling the role of immunophilin signaling in male fertility will help identify new targets for male contraceptives and/or alleviate male infertility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sperm maturation requires passage through the androgen-responsive epididymis. The epididymis is composed of three regions, the caput, corpus, and cauda, and each region contributes to specific functions, such as maturation, transport, concentration, and storage of sperm. Through maturation, sperm acquire motility and competence to undergo capacitation, physiological changes that culminate in sperm’s capacity to interact with and fertilize an oocyte (Orgebin-Crist 1967, Cornwall & Hsia 1997, Robaire & Hermo 2002, Nixon et al. 2005). The molecular mechanisms underlying these processes, however, are not completely understood.

Normal functioning of nuclear steroid hormone receptors is dependent on interactions with the molecular chaperone machinery to maintain a functional state competent for hormone binding and subsequent transcriptional activation (reviewed in Pratt & Toft 2003). Functionally mature steroid receptor complexes consist of a receptor monomer, a 90 kDa heat shock protein (HSP) dimer, the co-chaperone p23, and one of the four HSP co-chaperones that contain a tetratricopeptide repeat (TPR) domain (Smith 2004). The TPR co-chaperones include two members of the FK506-binding family of immunophilins FKBP52 and FKBP51, a member of the cyclosporin-binding immunophilin family cyclophilin 40, and the protein phosphatase PP5. FKBP52, FKBP51, and cyclophilin 40 are peptidylprolyl isomerases (PPIase), and they can influence conformation of protein substrates. While roles for HSPs in initiating and maintaining receptor competency for hormone binding are well documented, less is known about the contribution of other co-chaperones in receptor complexes. There is evidence that FKBP52 potentiates the function of glucocorticoid receptors (Riggs et al. 2003, Davies & Sanchez 2005, Wochnik et al. 2005), progesterone receptors (PR; Tranguch et al. 2005), and androgen receptors (AR; Cheung-Flynn et al. 2005). Although several studies suggest that FKBP52 plays a role in nuclear transport of receptor complexes, it is not clear whether this role is physiologically critical (Riggs et al. 2003, Cheung-Flynn et al. 2005, Davies & Sanchez 2005).

The infertility phenotype of both Fkbp52 null male and female mice on a C57BL/6/129 background demonstrates the critical function of this immunophilin co-chaperone in reproductive processes (Cheung-Flynn et al. 2005, Tranguch et al. 2005). We have recently shown that FKBP52 is a critical co-chaperone for uterine PR function during early pregnancy, and females missing the Fkbp52 gene show implantation failure (Tranguch et al. 2005). Fkbp52 null males exhibit numerous reproductive defects consistent with androgen insensitivity, including ambiguous external genitalia and dysgenic prostate (Cheung-Flynn et al. 2005). This is consistent with the findings that FKBP52 enhances AR-mediated transactivation in cellular models (Cheung-Flynn et al. 2005), and the loss of this FKBP52-enhancing activity accounts for major phenotypic features in Fkbp52 null males. While FKBP52 is expressed in most cell types in wild-type testes, testicular histology and spermatogenesis appear normal in null males (Cheung-Flynn et al. 2005). The abnormal external genital morphology of Fkbp52 null males prevents successful copulation; therefore, fertilization capacity has not yet been investigated in this mouse model. Here, we show that male mice missing FKBP52 on both C57BL/6/129 and CD1 backgrounds have abnormal sperm morphology and reduced capacity to fertilize wild-type oocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
The disruption of the Fkbp52 gene was achieved by homologous recombination as described previously (Cheung-Flynn et al. 2005). Tail genomic DNA was used for genotyping by PCR. Experiments were conducted on Fkbp52+/+ and Fkbp52–/– males on each genetic background (C57BL/6/129 and CD1) between 12 and 16 weeks of age. We introduced Fkbp52 deficiency in CD1 mice by crossing with C57BL/6/129 Fkbp52+/– mice. In brief, C57BL/6/129 Fkbp52+/– females were crossed to CD1 wild-type males producing an F1 generation. F1 Fkbp52+/– females were then backcrossed to CD1 wild-type males, and the process was continued for ten generations. All mice were housed in accordance with the National Institutes of Health (NIH) and Institutional guidelines on the care and use of laboratory animals.

In situ hybridization
In situ hybridization was performed as described previously by us (Das et al. 1994). In brief, frozen sections (10 µm) were mounted onto poly-L-lysine coated slides and fixed in 4% paraformaldehyde in PBS. The sections were prehybridized and hybridized at 45 °C for 4 h in 50% formamide hybridization buffer containing 35S-labeled antisense or sense cRNA probes. Probes had specific activities of approximately 2x109 d.p.m./µg. RNase A-resistant hybrids were detected by autoradiography. Sections were post-stained with hematoxylin and eosin. Sections hybridized with sense probes showed no positive signal and served as negative controls.

Immunohistochemical staining
Immunolocalization of AR and FKBP52 was performed in Bouin’s fixed paraffin-embedded sections as described previously (Cheung-Flynn et al. 2005, Daikoku et al. 2005).

Indirect immunofluorescence
Epididymal sperm were fixed on glass slides in 2% paraformaldehyde on ice. After washing, they were incubated in 8% BSA at room temperature for 1 h, exposed to FKBP52 antibody (1:200, custom-made) or propidium iodide (PI; 10 µg/ml; Sigma) for 16 h at 4 °C, washed and incubated with fluorescein isothiocyanate (FITC)-conjugated affinity purified donkey anti-rabbit IgG (FKBP52; 1:200; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Signals were captured undera fluorescent microscope (Nikon Microsystems, Melville, NY, USA).

Semi-quantitative RT-PCR
Total RNA was extracted from whole epididymis or individually from isolated caput, corpus, and cauda of wild-type and Fkbp52 null mice using Trizol reagent according to the manufacturer’s instructions. RT with oligo dT primers was performed to generate cDNAs from 5 µg total RNA using Superscript II. DNA amplification was carried out with Taq DNA polymerase (Invitrogen) using the following primers: Fkbp51 (403 bp), 5'-AAGGTGTTGGCAGTCAATCC-3', and 5'-GGTGGT CATTTGGGAAGCTA-3'; Adam7 (363 bp), 5'-GGTCATT GTGCTTGTCATGC-3', and 5'-ACGGAGGATAGCCCA GTCT-3'; Gpx5 (339 bp), 5'-AGCCAGCTATGTGCAG ACAA-3', and 5'-AACCCTTTTCCTGGACGAAC-3'; carbonic anhydrase 2 (Car2; 378 bp), 5'-ACCACT GGGGATACAGCAAG-3', and 5'-CCCCATATTTGGT GTTCCAG-3'; Serpine2 (357 bp), 5'-GGGATCCAGGTC TTCAATCA-3', and 5'-GATGGACTCAGAGGCAGAGG-3'; rPL7 (246 bp), 5'-TCAATGGAGTAAGCCCAAAG-3', and 5'-CAAGAGACCGAGCAATCAAG-3'. PCR conditions were 95 °C for 5 min and then 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s, followed by incubation at 72 °C for 10 min. Amplified fragments were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. The intensity of each band was measured by Scion Image (Scion Corp., Maryland, USA), and signals for Fkbp51, Adam7, Gpx5, Car2, and Serpine2 were evaluated by comparing against the intensity of a house-keeping gene rPL7.

Western blot analysis
Sperm (~106 cells) were isolated from the epididymis of mature wild-type males and thoroughly washed in PBS. Sperm were then pelleted by centrifugation at 3300 g for 5 min and resuspended in 250 µl SDS sample buffer. Supernatants were boiled for 5 min in SDS sample buffer containing ß-mercaptoethanol, run on 10% SDS-PAGE gels under reducing conditions and transferred onto nitrocellulose membranes. Membranes were blocked with 10% milk in Tris-buffered saline-Tween 20 (TBST) for 1 h at room temperature and then incubated in 1% milk containing anti-FKBP52 antibodies (1:5000) or anti-actin antibodies (1:500; Invitrogen) overnight at 4 °C. After incubation, membranes were washed thrice (15 min each) with TBST, incubated with goat anti-rabbit (FKBP52) or rabbit anti-goat (actin) IgG conjugated with horseradish peroxidase (1:10 000; Invitrogen) in 1% milk at room temperature, and washed thrice (15 min each) with TBST. The bands were detected using an enhanced luminescence kit (Amersham Pharmacia Biotech).

Sperm counts
Epididymis were isolated from mature Fkbp52+/+ or Fkbp52–/– males and placed into 500 µl preincubated human tubal fluid (HTF) medium (Specialty Media, Phillipsburg, NJ, USA). Sperm were collected by teasing the whole epididymis, and aliquots (20 µl) of a 1:10 dilution of this whole epididymal sperm suspension were counted using a hemocytometer. For morphological analysis, sperm were collected from each respective epididymal region and placed into HTF medium for 10 min at 37 °C for morphology observation under phase contrast microscopy.

Acrosome reaction
Sperm isolated from Fkbp52+/+ and Fkbp52–/– cauda epididymis were capacitated for 1.5 h followed by 5-min incubation in 10 µM calcium ionophore A23187 [GenBank] (Sigma). Dimethylsulfoxide (0.01%) was used to dissolve the ionophore and served as a vehicle control. Sperm were fixed in ice-cold 100% ethanol, air-dried onto poly-L-lysine coated slides, and labeled with tetra-methylrhodamine isothiocyanate (FITC)-conjugated Arachis hypogaea lectin (0.5 mg/ml in PBS, Sigma) at room temperature for 20 min, washed and mounted. Sperm were scored based on acrosomal reaction.

In vitro fertilization
In vitro fertilization was performed as described previously by us (Matsumoto et al. 2001). Briefly, wild-type C57BL/6/129 or CD1 female mice were super-ovulated by i.p. injections of 5 IU pregnant mare serum gonadotropin (Sigma) followed by injections of 5 IU human chorionic gonadotropin (hCG, Sigma) 48 h later. Cumulus–oocyte complexes were collected from the oviduct ampulla 12–14 h post-hCG injection and placed in 100 µl droplets of HTF medium (Chemicon, Temecula, CA, USA). Sperm were collected from whole epididymis of 8–12-week-old Fkbp52+/+or Fkbp52–/– males on C57BL/6/129 or CD1 backgrounds and placed in 400 µl HTF medium to allow capacitation for 2.5 h in a humidified 5% CO2 incubator at 37 °C. Sperm (~1.2–1.5x106 sperm/ml) were then co-incubated with oocytes to allow fertilization to occur. After 6 h, sperm were removed and putative zygotes placed in 30 µl drops of KSOM (potassium simplex optimized medium; Chemicon) and incubated in a humidified 5% CO2 incubator at 37 °C. The cleavage rate (two-cell stage) at 24 h was used as an index of fertilization. Formation of two-cell embryos to blastocysts at 120 h indicated the developmental potential of the fertilized embryos.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
FKBP52 is expressed in wild-type epididymis
For spermatozoa to become completely mature, they must pass through the epididymis, beginning their maturation process in the caput and finalizing maturation in the cauda region (Mathieu et al. 1992). The epididymis is responsive to androgen which helps to mature and concentrate spermatozoa, convert testosterone to 5{alpha}-dihydrotestosterone and to store spermatozoa (Inano et al. 1969, Setchell et al. 1993, Toshimori 2003). It has recently been shown that Fkbp52–/– males on a C57BL/6/129 background are infertile due to partial androgen insensitivity with ambiguous external genitalia, implying a role for FKBP52 in mediating functions of androgen-responsive tissues (Cheung-Flynn et al. 2005). FKBP52 expression has been detected in most spermatogenic cells of wild-type testes (Cheung-Flynn et al. 2005), but its expression has not yet been examined in the epididymis. To determine the cell specific expression of FKBP52 in wild-type epididymis, in situ hybridization and immunohistochemistry were performed. In situ hybridization detected Fkbp52 expression in epithelial cells of the initial segment (IS), caput, corpus, and cauda regions of the epididymis (Fig. 1AGo), albeit at low levels in the cauda. Localization of FKBP52 protein in similar cell types and regions confirms that the mRNA is effectively translated in the epididymis (Fig. 1BGo). FKBP52 protein was not detected in corresponding tissues of Fkbp52–/– mice (data not shown).


Figure 1
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  		Figure 1 Expression of FKBP52 in the epididymis. (A) In situ hybridization of Fkbp52 in different regions of the epididymis in wild-type and Fkbp52–/– mice was compared. Darkfield photomicrographs of representative sections of wild-type and Fkbp52–/– epididymis are shown (bar, 200 µm). (B) Immunolocalization of FKBP52 in wild-type epididymis. Brightfield photomicrographs of representative sections of wild-type epididymis are shown (bar, 100 µm). Red color depicts positive signals. IS, initial segment.

 
AR expression is normal in Fkbp52 null epididymis
FKBP52 is a known component of functionally mature steroid receptor complexes and potentiates AR function (Cheung-Flynn et al. 2005). Since immunostaining previously showed that AR expression is similar between wild-type and null testes (Cheung-Flynn et al. 2005), we compared AR expression in epididymis of wild-type versus Fkbp52–/– males. The pattern of AR localization in the epididymis was similar between wild-type and Fkbp52–/– mice. Specifically, AR staining was detected in nuclei of epithelial cells of the IS, caput, corpus, and cauda epididymis, showing that loss of FKBP52 does not alter AR expression or nuclear localization (Fig. 2AGo).


Figure 2
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  		Figure 2 Immunolocalization of androgen receptor (AR) and expression of androgen-regulated epididymal genes in Fkbp52+/+ and Fkbp52–/– males. (A) AR immunolocalization in wild-type and Fkbp52–/– epididymis. Brightfield photomicrographs of representative sections of wild-type and Fkbp52–/– epididymis are shown (bar, 200 µm). IS, initial segment. (B) Semi-quantitative comparative RT-PCR of androgen-regulated genes (Adam7, Gpx5, Car2, Serpine) and Fkbp51 in wild-type and Fkbp52–/– mice. rPL7 is a housekeeping gene. (C) RT-PCR data are presented as fold changes (mean±S.D.) of three independent RNA samples (P>0.05; unpaired t-test).

 
Androgen-responsive genes are normally expressed in Fkbp52 null epididymis
We have recently shown that Fkbp52–/– females with compromised PR function maintain normal levels of PR (Tranguch et al. 2005), confirming FKBP52’s role in determining PR activity rather than affecting its expression levels. Our finding of normal AR expression in Fkbp52–/– epididymis suggests that FKBP52 is not required for accumulation or nuclear localization of AR, but is critical for receptor activity. If AR functions are compromised in Fkbp52–/– epididymis, then androgen-regulated genes would be aberrantly expressed. We selected androgen-regulated genes that encode a disintegrin metalloprotease 7 (Adam7; Cornwall & Hsia 1997), glutathione peroxidase 5 (Gpx5; Schwaab et al. 1998), Car2 (Kaunisto et al. 1999), and Serpine2 (Vassalli et al. 1993). Whereas Adam7 has been previously characterized as an androgen-regulated gene in the epididymis (Zwain et al. 1992, Cornwall & Hsia 1997), Gpx5, Car2, and Serpine2 are androgen-regulated genes expressed specifically in the caput, corpus, and cauda respectively (Chauvin & Griswold 2004). The whole epididymis, and the caput, corpus, and cauda regions were isolated from wild-type and Fkbp52–/– males. Semi-quantitative RT-PCR shows that the expression of Adam7, Gpx5, Car2, and Serpine2 is similar between wild-type and Fkbp52–/– males in the whole epididymis or in specific epididymal regions (Fig. 2B and CGo and data not shown). We compared the levels of these genes with a housekeeping gene rPL7 run under the same condition.

FKBP52 and FKBP51 compete for a common binding site on HSP and thus for assembly with steroid receptor complexes (Riggs et al. 2003). Furthermore, FKBP51 has been shown to antagonize the action of FKBP52 on steroid hormone receptor function (Riggs et al. 2003). It is surprising that functional differences exist between these two immunophilins given the 70% homology in their amino acid sequences (Nair et al. 1997). For example, while the infertile phenotype of Fkbp52 null males implicates its important role in fertility, Fkbp51 null males apparently do not display any reproductive defects (Cheung-Flynn et al. 2005). To determine whether Fkbp51 is overexpressed in Fkbp52–/– males to produce the observed infertility phenotype, we used comparative RT-PCR and found that Fkbp51 expression is not altered in Fkbp52–/– epididymis (Fig. 2B and CGo). These results indicate that the infertile phenotype observed in Fkbp52–/– males is specific to Fkbp52 deficiency, and not due to aberrant expression of Fkbp51.

FKBP52 plays a role in governing sperm morphology
We observed FKBP52 presence in spermatozoa of wild-type epididymis (Fig. 1A and BGo). This observation was further confirmed by western blotting detection of FKBP52 protein in sperm isolated from wild-type epididymis (Fig. 3AGo). To determine FKBP52 localization in wild-type sperm, we performed indirect immunofluorescence and found its expression specifically in the acrosome of sperm, and in the midpiece and annulus of sperm tails (Fig. 3BGo). The annulus is located at the distal end of the midpiece and is thought to serve as a stabilizing structure for tail rigidity (Cesario & Bartles 1994). Indeed, annulus-deficient sperm have been shown to exhibit an abnormal bent morphology (Kissel et al. 2005). A similar bent morphology is observed in Fkbp52–/–sperm (Fig. 4AGo). In fact, almost 40% of cauda epididymal sperm had abnormal flagella morphology, forming hairpin bends (Fig. 4BGo). Sperm isolated from the caput and corpus regions of the epididymis, however, did not show abnormal morphology (Fig. 4BGo).


Figure 3
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  		Figure 3 Expression of FKBP52 in sperm. (A) Western blot analysis of FKBP52 in wild-type epididymal sperm. Pregnant day-4 uterus served as a positive control. Actin is a housekeeping protein. (B) Indirect immunofluorescence of wild-type and Fkbp52–/– sperm with anti-FKBP52 (FITC, green) antibodies. Sperm head DNA was stained with propidium iodide (PI; red). While white arrowheads denote FKBP52 localization specifically in the midpiece and annulus of sperm tails, yellow arrows denote FKBP52 localization in the sperm acrosome (bar, 100 µm).

 

Figure 4
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  		Figure 4 Sperm morphology in wild-type versus Fkbp52–/– mice. (A) Sperm isolated from the cauda epididymis were stained with hematoxylin and eosin. A representative brightfield photomicrograph is shown (bar, 100 µm). Arrowheads denote hairpin bends frequently observed in sperm of Fkbp52–/– males. (B) Sperm morphology was analyzed from the caput, corpus, and caudal regions of wild-type and Fkbp52–/– epididymis. Abnormal sperm morphology (hairpin bends) in Fkbp52–/– males was only observed in sperm isolated from the cauda epididymis (*P<0.01; unpaired t-test). Data are presented from 20 (Fkbp52+/+) and 22 (Fkbp52–/–) independent samples. (C) Sperm count is significantly lower in Fkbp52–/– epididymis when compared with wild-type males (*P<0.05; unpaired t-test). Data are presented from 6 Fkbp52+/+ and Fkbp52–/– male mice on each genetic background.

 
Fkbp52 null sperm exhibit reduced fertilization capacity
To address whether spermatogenesis occurs normally in Fkbp52–/– males, sperm were collected from wild-type and Fkbp52–/–epididymis and counted. The number of sperm collected from the whole epididymis was significantly lower in Fkbp52–/–males when compared with wild-type littermates on both genetic backgrounds (Fig. 4CGo). This observation was not surprising since androgen receptor signaling is known to govern maintenance of spermatogenesis (Keller et al. 1996, Roy et al. 1999, Heinlein & Chang 2002). To address whether inherent defects in sperm also contribute to male infertility, we first selected Fkbp52–/– males with minimal penile dysgenesis for breeding with wild-type females. However, we failed to detect any vaginal plugs from these breedings (data not shown). This may suggest poor mating activity of Fkbp52–/– males. We then sought to determine whether Fkbp52–/– sperm are capable of fertilizing wild-type oocytes. We performed in vitro fertilization using isolated sperm from Fkbp52–/– males on either C57BL/6/129 or CD1 background with eggs isolated from wild-type females on the same genetic background respectively. Sperm isolated from wild-type C57BL/6/129 males exhibited a 76% fertilization rate with wild-type eggs with 91% of fertilized embryos developing to the blastocyst stage (Fig. 5A and BGo). On the other hand, sperm isolated from C57BL/6/129 Fkbp52–/– males showed a markedly reduced fertilization rate (44%). However, 88% of those fertilized embryos developed to the blastocyst stage which is comparable with rates seen for embryos fertilized by wild-type sperm (Fig. 5A and BGo).


Figure 5
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  		Figure 5 In vitro fertilizing capacity of wild-type and Fkbp52–/– sperm. (A) Percentage of wild-type oocytes fertilized by wild-type or Fkbp52–/– sperm on either C57BL/6/129 or CD1 background. Fertilizing capacity of Fkbp52–/– sperm is significantly lower than wild-type sperm (*P<0.01; {chi}2-analysis). Numbers above bars indicate the sum of number of fertilized oocytes/total oocytes for all three experiments. (B) Percentage of fertilized embryos (two-cell) resulting from wild-type and Fkbp52–/–sperm that developed to blastocysts. No difference was observed between wild-type and Fkbp52–/– mice on either genetic background. Numbers above bars indicate the sum of fertilized oocytes developed to the blastocyst stage/total fertilized oocytes for all three experiments. For each experiment, two males of each genotype were used, and oocytes were collected from eight CD1 or ten C57BL/6/129 wild-type females. Each experiment was performed thrice. (C) In vitro acrosome reaction of caudal epididymal sperm from wild-type and Fkbp52–/– males. Representative brightfield (left panel) and darkfield (right panel) photomicrographs of sperm isolated from Fkbp52–/– males (bar, 100 µm). The acrosome reaction is denoted by green staining of FITC-conjugated Arachis hypogaea lectin. Arrows denote sperm that completed the acrosome reaction. Sperm heads were stained using propidium iodide (red). (D) The percentage of sperm showing acrosome reaction was comparable between wild-type and Fkbp52–/–males (P>0.05; unpaired t-test). 0 h, sperm without incubation/treatment; 1.5 h, incubation at 37 °C post-extraction; DMSO, incubation at 37 °C post-extraction (1.5 h)+DMSO treatment (0.01%); A23187, incubation at 37 °C post-extraction (1.5 h)+calcium ionophore (10 µM). DMSO was used to dissolve the calcium ionophore A23187. Experiments were performed in triplicate. Numbers above (0 h) and within bars indicate acrosome-reacted sperm/total sperm.

 
There is increasing evidence that mutation of a gene often results in substantially altered phenotypes depending on the genetic background of mice in which the mutation is maintained. It was previously noted that abnormalities in sexual development, including external and internal genitalia, in Fkbp52 null males on both C57BL/6/129 and CD1 background is similar (Cheung-Flynn et al. 2005). Similar to C57BL/6/129 Fkbp52–/– males as described earlier, we found that CD1 Fkbp52–/– males bred with wild-type females failed to produce vaginal plugs (data not shown). In fact, the infertility phenotype was maintained in CD1 Fkbp52–/– males. This prompted us to examine the fertilizing capacity of sperm from CD1 Fkbp52–/– mice in vitro. We found that sperm isolated from wild-type CD1 males show normal fertilization (93%) of wild-type eggs with 90% of fertilized embryos developing to blastocysts (Fig. 5A and BGo). In contrast, sperm isolated from CD1 Fkbp52–/– males again showed compromised fertilization rate (73%), although development of fertilized embryos to blastocysts proceeded normally; 88% of two-cell embryos developed to blastocysts (Fig. 5A and BGo). The in vitro fertilizing capacity of sperm from CD1 Fkbp52–/– males was not as dramatic as that of C57BL/6/129 Fkbp52–/– males. This is not unexpected, since outbred CD1 mice have more robust reproduction than those on a C57BL/6/129 background. The observed reduction in fertilization capacity in Fkbp52 null mice on both genetic backgrounds provides convincing evidence for a role for FKBP52 in fertilization.

It is known that the failure of the acrosome reaction can contribute to sperm’s fertilization capacity (Sabeur et al. 1996). Because of our observation of FKBP52 localization in sperm acrosome (Fig. 3BGo), we tested whether the acrosome reaction occurs normally in Fkbp52–/– sperm by examining the response of wild-type and Fkbp52 null sperm to a calcium ionophore A23187. [GenBank] No significant differences in the induction of acrosome reaction were noted between wild-type and Fkbp52–/– sperm (Fig. 5C and DGo), suggesting an alternative role for FKBP52 in mediating sperm–egg interactions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Although FKBP52 is expressed in a wide variety of vertebrate tissues (Peattie et al. 1992, Nair et al. 1997), defects in Fkbp52–/–males are apparently restricted to the reproductive organs (Cheung-Flynn et al. 2005). The reproductive phenotype of Fkbp52 null male mice results from partial androgen insensitivity in a selected number of reproductive organs, specifically the anterior prostate and external genitalia (Cheung-Flynn et al. 2005). While the epididymis is an androgen-responsive tissue, the finding of normal development of the epididymis and expression of androgen-regulated genes in the epididymis in Fkbp52–/– males is surprising. It is possible that locally high androgen levels in epididymal tissue lessen the need for FKBP52 in AR complexes (Cheung-Flynn et al. 2005). Nonetheless, our observations of localization of FKBP52 in wild-type spermatozoa and compromised in vitro fertilizing capacity of Fkbp52–/– sperm indicate a novel role for this immunophilin co-chaperone in sperm function.

Biogenesis of mammalian sperm tails begins early during spermatogenesis with the development of a primary flagellum, a simple axoneme enveloped by a plasma membrane (Irons & Clermont 1982). Proteins involved in flagellar maturation are assumed to be synthesized in the spermatid cell body and transported down the axoneme to sites of assembly (Irons & Clermont 1982). FKBP52 expression specifically in the midpiece and annulus of spermatozoa, and abnormal Fkbp52–/– sperm morphology implicates a role for FKBP52 in tail development and/or movement. FKBP52 binds to dynein, and evidence suggests direct binding to dynein through FKBP52’s PPIase domain (Silverstein et al. 1999, Galigniana et al. 2001). FK506, an immunosuppressant drug, binds to the PPIase active site to block PPIase activity, although FKBP52 binding to dynein in vitro is not inhibited by FK506 (Silverstein et al. 1999). Still, FK506 has been shown to reduce sperm counts and motility in a rat model (Hisatomi et al. 1996), suggesting FKBP52-dynein interactions in vivo. Consistent with a possible role for FKBP52–dynein interactions in sperm flagella, mice lacking functional dynein heavy chain (Dnahc1) show severe asthenozoospermia (Neesen et al. 2001, Vernon et al. 2005). It is thus possible that FKBP52 functions independent of steroid receptor, perhaps interacting with dynein to govern flagella formation and/or maturation for successful fertilization.

Sperm–egg interactions involve binding of acrosome-intact spermatozoa to the zona pellucida, induction of the acrosome reaction, consequent binding of acrosome-reacted sperm to the zona pellucida matrix, penetration through the matrix, and binding to the oocyte plasma membrane for sperm–oocyte fusion (Nixon et al. 2005). The role of chaperones and co-chaperones in sperm–egg recognition and interaction remains unclear. It has been shown that mammalian sperm express surface chaperones including endoplasmin, HSP60, HSP70, and HSP90 that each could play roles in interacting and binding to the zona pellucida (Bohring et al. 2001, Bohring & Krause 2003, Ecroyd et al. 2003, Ficarro et al. 2003). FKBP52’s classification as a HSP90-binding immunophilin and perhaps its ability to bind with other heat shock protein family members suggests a role for FKBP52 during these events. But the exact mechanism by which FKBP52 mediates sperm–egg interactions during fertilization remains to be determined. Recently, several rapid non-genomic effects of progesterone and estrogen have been described for human spermatozoa (Baldi et al. 1998, 2000). These effects include, among others, calcium influx, tyrosine phosphorylation of various substrates, and increased cAMP levels, all contributing to capacitation and hyperactivated motility of sperm (Baldi et al. 1998, Luconi et al. 2004). Whether FKBP52 participates in membrane steroid hormone receptor signaling remains to be determined. Nonetheless, the present study provides evidence for roles of FKBP52 in sperm morphology and fertilizing capacity. This presents a clinically relevant finding since FKBP52 can perhaps serve as a target for developing novel contraceptives and for treatment of male infertility.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Hiromichi Matsumoto for his assistance with in vitro fertilization, Toshifumi Takahashi for assistance with immunofluorescence experiments and Fuhua Xu for help with statistical analysis. We also thank Carlos Suarez-Quian for critical reading of the manuscript. This work was supported in parts by NIH grants (HD 12304 and DA06668). ST is supported by NIH grant 5 T 32 DK07563. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 29 August 2006
First decision 27 September 2006
Accepted 9 November 2006

J Hong and S T Kim contributed equally to this work

S T Kim is now at Department of OB-GYN, Washington University, St Louis, Missouri, USA


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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