# Phospholipid transfer protein (PLTP) deficiency reduces sperm motility and impairs fertility of mouse males

Véronique Drouineaud, Laurent Lagrost, Alexis Klein, Catherine M

Desrumaux, Naig Le Guern, Anne Athias, Franck Ménétrier, Philippe

Moiroux, Paul Sagot, Clément Jimenez, et al.

► To cite this version:

Véronique Drouineaud, Laurent Lagrost, Alexis Klein, Catherine M Desrumaux, Naig Le Guern, et al. Phospholipid transfer protein (PLTP) deficiency reduces sperm motility and impairs fertility of mouse males. FASEB Journal, 2006, 20 (6), pp.794 - 796. 10.1096/fj.05-5385fje. hal-04203338

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# Phospholipid transfer protein (PLTP) deficiency reduces sperm motility and impairs fertility of mouse males.

Véronique Drouineaud,* Laurent Lagrost,†‡ Alexis Klein,†‡ Catherine Desrumaux,†‡
Naig Le Guern,†‡ Anne Athias,†‡ Franck Ménétrier,‡ Philippe Moiroux,* Paul Sagot,§
Clément Jimenez,* David Masson,†‡ and Valérie Deckert†‡

*Laboratoire de Biologie de la Reproduction, EA Génétique et Reproduction, Dijon, France;
†INSERM U498, Faculté de Médecine, Dijon, France; ‡IFR 100, Faculté de Médecine, Dijon,
France; and §Service de Gynécologie Obstétrique, Hôpital du Bocage, Dijon, France

Corresponding authors: Laurent Lagrost or Valérie Deckert, INSERM U498, Faculté de Médecine, 7 Bd Jeanne d'arc, BP87900, 21079 Dijon Cedex France. E-mail: laurent.lagrost@ubourgogne.fr; valerie.deckert@ubourgogne.fr

## ABSTRACT

Vitamin E was discovered for its implication in reproductive biology, and its transport in mammalian plasma and brain was shown to be governed by plasma phospholipid transfer protein (PLTP). We show that PLTP deficiency is associated with hypofertility of mouse males but not mouse females, and it accounts for a significant decrease in total number of pups produced over a 2-month breeding period of PLTP knocked out mice (–32%, *P*<0.03). PLTP is highly expressed in epididymis of mouse males, and α-tocopherol, the main vitamin E isomer in vivo, was significantly less abundant in cauda and caput epididymis of PLTP-deficient mice as compared with wild-type counterparts (caput: –26%, *P*<0.05; cauda: –21%, *P*<0.05). Mature spermatozoa from PLTP-deficient epididymis were shown to retain an abnormal α-tocopherol content. PLTP deficiency tended to reduce sperm motility as shown by a 24% reduction in spermatozoa with progressive motility (*P*<0.02), with no change in other sperm parameters as compared with wild-type males. Finally, in vitro fertilization rates of wild-type oocytes with spermatozoa from PLTP-deficient males were markedly reduced as compared with those measured with spermatozoa from wild-type males (–60%, *P*<0.05). It is concluded that PLTP is a new, key factor that determines sperm motility and male fertility.

Key words: alpha-tocopherol • vitamin E • reproductive biology

Phospholipid transfer protein (PLTP) was first described as a plasma factor that facilitates the exchange of phospholipid species between circulating lipoproteins (1, 2). In addition, PLTP can bind and transfer several other amphipatic compounds, including unesterified cholesterol, lipopolysaccharides, diacylglycerides, and vitamin E (2). PLTP can influence vascular biology through its ability to modify structure, composition, production, and antioxidative protection of circulating lipoproteins. Briefly, PLTP deficiency in hyperlipidemic mice leads to significant reduction in atherosclerosis susceptibility, which has been explained in terms of a combination of a significant blockade of the production of atherogenic apoB-
containing lipoproteins by the liver (3) and a significant rise in their antioxidative protection (4). Accordingly, PLTP overexpression in genetically engineered mice led to a rise in atherosclerotic lesions as a result of increased circulating levels and reduced antioxidative protection of atherogenic apoB-containing lipoproteins (5). These abnormalities were proven to result at least in part from abnormalities in the liver (6) and intravascular (5) distribution of α-tocopherol, i.e., the major vitamin E isomer in vivo. Recently, PLTP deficiency was also proven to display anti-inflammatory properties, an observation that comes in further support of the proatherogenic effect of PLTP in hyperlipidemic state (7, 8).

Beyond its impact on vascular biology, the vitamin E transfer activity of PLTP might well produce a number of other biological effects. For instance, PLTP is expressed at elevated levels in mouse brain, a major vitamin E reservoir that was recently found to rely in part on active PLTP for normal incorporation and distribution of vitamin E (9). PLTP deficiency leads to fewer α-tocopherol molecules in mouse brain, a phenotype that produces lower antioxidative protection of brain lipids together with an anxious behavior (9). Interestingly, testis constitutes another organ that is known to rely tightly on vitamin E for normal function. Vitamin E was first discovered as a major determinant of male fertility (10). In addition, testis, which displays high expression level of PLTP (11), also displays elevated vitamin E turnover (12). All these observations raised the important question of the physiological impact of PLTP expression in reproductive biology.

In the present study, the effect of PLTP on reproductive biology was investigated through the comparison of wild-type and PLTP-knocked out mice. A significant hypofertility was found in PLTP-deficient males, with no significant alteration of fertility in PLTP-deficient females. As compared with other tissues, epididymis expresses elevated levels of PLTP in wild-type males, and concordant observations led to the conclusion that the motility of vitamin E-deficient spermatozoa from PLTP-deficient mouse males is significantly decreased. Resulting reduction in fertility was assessed by a significant decrease in in vitro fertilization rate when normal oocytes from wild-type, superovulated mouse females were incubated in the presence of PLTP-/-spermatozoa.

# MATERIALS AND METHODS

## Animals

PLTP-deficient (PLTP-/-) mice (kindly provided by Dr. X.C. Jiang and Dr. A.R. Tall, 13) and wild-type mice in a homogenous C57BL/6 background were fed a standard chow diet (A03 diet, SAFE, France—vitamin E content, 30 mg per kilogram) ad libitum. Experiments involving animals were approved by the Ethical Committee on the Use of Laboratory Animals of the University of Burgundy and they were performed in accordance with institutional guidelines.

## Breedings-fertility assessment

To compare fertility of PLTP-/- mice and wild-type (WT) mice, 1-4 month old, virgin females were continuously mated with 2-4 month-old males in individual cages. Four types of pairs were formed: WT males with WT females (n=16); PLTP-/- males with PLTP-/- females (n=18); WT
males with PLTP-/- females (n=5), and PLTP-/- males with WT females (n=7). The numbers of litters and pups were counted over a 2-month mating period.

## Tissue collection

Animals (12 to 66 weeks old) were killed by intraperitoneal injection of sodium pentobarbital and blood intracardiac puncture. Blood samples were centrifuged and plasma stored at -80°C until measurements. Testis, caput, and cauda epididymis, vesicular glands, prostatis, and coagulating glands were removed and weighted. According to the subsequent determinations, organs were either used as fresh preparations for determination of concentration, motility, viability, morphology of spermatozoa, as well as for immunohistological analysis. Samples were stored at -80°C for subsequent testosterone and vitamin E assays.

## RNA isolation and real-time PCR

Total RNA was isolated from tissues with Trizol (Eurogentec, Seraing, Belgium). RNA levels were quantified by reverse transcription followed by real-time PCR using an ABI PRISM 7700 Sequence Detection System instrument (Applied Biosystems, Courtaboeuf, France). Reactions were performed by using the Quantitect SYBR Green amplification kit (Qiagen S.A., Courtaboeuf, France) following the instructions provided by the manufacturer. PLTP was normalized to the 18S internal control. Relative PLTP mRNA levels were calculated using the ΔΔ Ct method. Specific primer sequences were 5'-TCGGCGGAGGGTGTGTCCAT-3' (sense) and 5'-CATGGCAGAGTCAAAGAAGA-3' (antisense) for mouse PLTP, and 5'-GGGAGCCTGAGAAACGGC-3' (sense) and 5'-GGGTCGGGAGTGGGTAATTT-3'(antisense) for 18S.

## Immunohistochemistry

Dissected epididymis were fixed by immersion in 4% paraformaldehyde for 24 h, placed in a sucrose solution, and then embedded in OCT (Optimal Cutting Compound) at -20°C. Epididymis were serially cut into 10 µm sections on a cryostat-microtome. Non-specific sites were blocked with a 10% goat serum for 30 min. After rinsing in PBS, sections were incubated for 16 h at room temperature in an humidified chamber with rabbit anti-PLTP antiserum (Abcam Ltd, Cambridge, UK) diluted 1/100 in PBS containing 0.3% Triton X100, 1% bovine serum albumin, and 0.01% sodium azide. Sections were then rinsed in PBS containing 0.3% Triton X 100 and incubated with a secondary antibody conjugated to FluoProbes (1/400, Molecular Probes-Interchim, France) for 1 h at room temperature.

## Histology

Dissected testes were fixed in Bouin solution for 24 h, processed, and paraffin-embedded using standard protocols. The initial morphology of the testis was examined after hematoxylin and eosin staining of 5 µm paraffin sections.

## Testosterone measurements

Testosterone was assayed in plasma and in testis homogenate by gas chromatography-mass spectrometry (GC-MS). Extraction was conducted as described previously for rat brain and
plasma (14). After passage through a C18 solid-phase column, recovered extract was dissolved in 200 µl of acetonitrile and 20 µl of heptafluorobutyric anhydride (Sigma), and it was incubated for 30 min at room temperature and evaporated to dryness. The extract was finally dissolved in 50 µl of hexane. Quantitation of testosterone was performed using a HP-5MS fused silica capillary 30 m × 0.25 mm column (Agilent Technologies) in a Hewlett Packard HP6890 gas chromatograph equipped with an HP7683 injector and a HP5973 mass selective detector operated in the EI mode. The GC-MS conditions were as follows: carrier gas, helium at a flow rate of 1.1 mL/min; injector temperature, 250°C; oven temperature 150°C, increased at 15°C/min to 300°C and held for 5 min. Ions at m/z 680 and 683 were used to measure testosterone and testosterone-d3, respectively. Testosterone level was determined by comparison with a standard curve that was obtained with known amounts of testosterone (Riedel-de-Haën 46923).

## Sperm parameters

Mouse males were individually housed for 5 days prior to be killed. Spermatozoa were collected in 500 µL prewarmed Ferticult (FertiPro, Beernem, Belgium) by cutting cauda epididymis, and they were allowed to disperse for 30 min at 37°C.

Sperm concentration was determined with a hematocytometer on a sample of the epididymal sperm suspension. To analyze sperm morphology, smears were prepared and a Schorr and Harris' hematoxylin staining procedure was used. Percentages of morphologically normal and abnormal spermatozoa were evaluated on 100 sperm cells at a final magnification of 1000. Abnormal mouse spermatozoa lack the usual hook, have a bent form, are amorphous, are folded on themselves, or possess two tails (15). Assessement of alive and dead spermatozoa was performed using an eosin-nigrosin blue staining mixture (16).

Sperm motility was determined by assessing at least five microscopic fields to classify 100 spermatozoa (×400 magnification). The motility was estimated by using the basic procedure established for routine human semen analyses (17) and graded progressive (fast and slow), non-progressive or immotile.

## In vitro fertilization

Wild type, 3-4 week old mouse females (n=10) were induced to superovulate by an intraperitoneal injection of pregnant mare serum gonadotropin (10 IU, Intervet, France) followed by an intraperitoneal injection of human chorionic gonadotropin (10 IU, Organon SA, France) 48 h later. Females were killed 13-15 h after hCG injection by an intraperitoneal injection of sodium pentobarbital and blood intracardiac puncture. Oviducts were collected and the cumulus-oocyte complexes were removed. Normal oocytes were transferred to 150 µl drops of human tubal fluid medium (HTF; Cambrex BioScience) supplemented with penicillin (0.75 µg/mL; Sigma), streptomycin (0.5 µg/mL; Sigma) and bovine serum albumin (0.4% (w/v); Sigma). Droplets were covered by embryo-tested mineral oil (Sigma) and incubated at 37°C in 5% CO2. Twenty-three to 30 week-old wild-type (n=5) and PLTP-/- mouse males (n=5) were caged alone at least five days before being killed. Spermatozoa collected from the cauda epididymis were allowed to swim into supplemented HTF medium and were then incubated for 2 h at 37°C in 5% CO2 to allow capacitation to occur and finally added to oocyte droplets to achieve a concentration of 106 cells per mL. In vitro fertilizations were performed for 5-7 h at 37°C and 5% CO2 after which the oocytes were carefully removed and washed three times with fresh HTF.
After 18 to 20 h of incubation at 37°C, eggs were examined for the presence of the two-cell-stage embryos as an indication of successfull fertilization. Sperm fertility (i.e., fertilization rate) was defined as the percentage of 2-cell embryos obtained.

## Alpha-tocopherol determinations

### Epididymal spermatozoa isolation for Vitamin E determinations

Epididymis was rapidly removed, and caput and cauda were separated and placed in 500 µL prewarmed Ferticult (FertiPro, Beernem, Belgium). Three gentle cuts in caput and cauda epididymis were performed with fine scissors to allow spermatozoa to disperse for 30 min at 37°C. Then ferticult containing spermatozoa in suspension was transferred to fresh tube for 5 min centrifugation at 1500 rpm and room temperature. Supernatant was discarded and spermatozoa were subsequently washed three times with 1 mL PBS containing 20 µM butylated hydroxytoluene (BHT). Spermatozoa from caput and cauda were re-suspended in 100 µL and 200 µL of PBS containing BHT, respectively. Cell counting was performed in a hematocytometer. Cell suspensions were stored at -80°C until vitamin E analysis.

### α-Tocopherol quantitation in tissue and spermatozoa

Alpha-tocopherol was assayed in tissue homogenates and spermatozoa suspensions by gas chromatography-mass spectroscopy (GC-MS). Samples were sonicated in ice with 1 mL of saline solution (NaCl 150 mM) containing 25 mM ascorbic acid. The extraction procedure was conducted as described previously (18). Briefly, 1 mL of an ethanolic solution containing 50 mg/L BHT, 300 µL of 10 M KOH, and 50 µL of 10 µg/mL ethanolic solution containing Tocol (Spiral, France) as an internal standard were added, and saponification was conducted at 80°C for 30 min with intermittent shaking. The saponified solution was cooled down on ice water, and it was mixed with 4 mL of hexane. The mixture was shaken for 1 h, and the upper layer was collected after low-speed centrifugation and evaporated. The extract was dissolved in 100 µL of a mixture of BSTFA [Bis(trimethylsilyl)trifluoroacetamide] and TMCS (trimethylcholrosylane) 4/1 v/v, incubated 45 min at 80°C and evaporated. The extract was finally dissolved in 100 µL of hexane. Quantitation of tocopherol was performed using a HP-5MS fused silica capillary 30 m × 0.25 mm column (Agilent Technologies) in a Hewlett Packard HP6890 Gas chromatograph equipped with an HP7683 injector and a HP5973 mass selective detector operated in the EI mode. The GC-MS conditions were as follows: carrier gas, helium at a flow rate of 1.1 mL/min; injector temperature, 250°C; oven temperature 150°C, increased at 15°C/min to 280°C, then at 2°C/min to 300°C, and held for 5 min. Ions at m/z 502 and 460 were used to measure tocopherol and tocol, respectively. Tocopherol level was determined by comparison with a standard curve that was obtained with known amounts of α-tocopherol (Fluka 89550).

## Statistics

Results are presented as mean ± SEM. The statistical significance of differences between data means was determined by using either Mann-Whitney *U* test or Wilcoxon signed rank test, as appropriate.
# RESULTS

## PLTP deficiency affects male, but not female fertility

To characterize the effect of PLTP deficiency on the reproductive function, a continuous breeding study was conducted in WT and PLTP-/- mice over a 2-month period. Day number to the first litter did not differ between PLTP-/- and wild-type mice [32.8±3.9 (n=18) vs 33.3±3.9 (n=16) days, respectively; ns], and the number of litters over the 2-month study period did not differ either (2.1±0.2 and 1.9±0.2 litters, respectively; ns). In contrast, the total number of pups produced over the breeding period was significantly reduced with PLTP-/- mouse-breeding pairs as compared with wild-type counterparts (-32%; P<0.03) (Fig. 1). When PLTP-/- males were crossbred with WT females, the total number of pups produced over the 2-month period was again decreased by 31% (P<0.05) as compared with wild-type pairs, whereas no difference was observed when PLTP-/- females were crossbred with WT males, indicating that PLTP deficiency is associated with hypofertility of mouse males only.

## Epididymis is one of the strongest PLTP expressors among tissues of mouse males

Although significant expression level of PLTP has been previously demonstrated in a number of mouse tissues, including lung, brain, heart, kidney, and testis (11), its expression pattern in male reproductive organs and accessory glands had never been specifically addressed. As shown in Fig. 2, PLTP exhibits a widespread distribution among reproductive organs of mouse males. Levels of PLTP mRNAs were markedly elevated in epididymis, in particular with tremendous, ~7-fold higher PLTP expression levels in caput epididymis as compared with testis or liver. Immunohistochemical analysis showed a strong PLTP staining in caput and cauda epididymis of WT mice, with no detectable signal in PLTP-/- mouse tissues (Fig. 3). Interestingly, the highest PLTP staining intensity was preferentially localized in the apical surface of epididymal principal cells in the caput region (Fig. 3).

## Spermatozoa from PLTP-/- mice are vitamin E-deficient

Reduced fertility of PLTP-/- mice did not appear to result from a spermatogenic defect, since light microscopy analysis showed no differences in the architecture of the testis from WT and PLTP-/- mice. In particular, seminiferous tubules of PLTP-/- mice appeared normal and all stages of spermatogenesis, including spermatogonia, spermatocytes, and spermatids, were present in the two groups (Fig. 4).

Beyond the morphological analysis, epididymis and spermatozoa were analyzed for their α-tocopherol content, which is well known to constitute a key factor in reproductive biology. This specific analysis was urged by recent data, which demonstrated that PLTP plays a determinant role in the distribution of α-tocopherol in several tissues, including blood (4), vascular wall (4), and brain (9). As shown in Table 1, α-tocopherol concentration was significantly lower in cauda and caput epididymis of PLTP-/- mice compared with wild-type counterparts (caput: -26%, P<0.05; cauda: -21%, P<0.05). Isolated spermatozoa from caput and cauda epididymis of PLTP-/- mice retained the abnormalities observed in the α-tocopherol content of epididymis (Table 1), and PLTP deficiency induced a significant decrease in the α-tocopherol content of
spermatozoa from PLTP-/-mice as compared with spermatozoa from wild-type mice (caput: -29%, P<0.01; cauda: -26%, P<0.05).

## Spermatozoa from PLTP-/- mice show reduced motility

Body weight did not differ between PLTP-/- and WT mice, and weights of testis, epididymis, prostatis, vesicular, and coagulating glands were identical in PLTP-/- and WT counterparts (Table 2). No significant difference in plasma and testicular testosterone levels were observed between wild-type and PLTP-/- mice (plasma: 2.27±0.75 and 2.78±0.85 ng/mL, respectively, n=9, ns; testis: 533±94 pg/mg and 466±51 pg/mg, respectively, n=11, ns). Concentration, viability, morphology, and motility of spermatozoa from cauda epididymis were analyzed. As shown in Table 3, spermatozoa from PLTP-/- mice exhibited normal concentration, viability, and morphology as compared with WT mice. However, PLTP deficiency led to fewer spermatozoa with progressive motility (-24%, P<0.02), corresponding to an overall, significant 9% decrease in observed progressive motility of sperm (P<0.02) as compared with wild-type mice (Fig. 5).

## In vitro fertilization rate of mouse oocytes by PLTP-/- spermatozoa is reduced

In vitro fertilization assays were performed to assess the ability of PLTP-/- spermatozoa to fertilize eggs. Mature eggs from superovulated wild-type females were incubated for 24 h with capacitated WT or PLTP-/- spermatozoa originated from the cauda epididymis. The appearance of two-cell-stage embryos was taken to indicate successful penetration of spermatozoa and subsequent activation of fertilization. As shown in Fig. 6, the fertilization rate with PLTP-/- spermatozoa was markedly reduced as compared with the fertilization rate measured in parallel with WT spermatozoa (-60%, P<0.05. Fig. 6).

## DISCUSSION

Approximately 60% of all cases of male infertility in humans are attributed to genetic factors, and the elucidation of the underlying gene defects is an area of intensive research (19, 20). In the present study, plasma phospholipid transfer protein (PLTP), i.e., a protein that is normally expressed at high levels in several mouse and human tissues, arose as a new candidate, which may play a crucial role in reproductive biology. PLTP deficiency is associated with a lower fertility of mouse males, but not mouse females. Spermatozoa motility is significantly decreased in PLTP-/- mouse males in the absence of morphological abnormalities. In vitro fertilization experiments indicated that PLTP-/- spermatozoa are less prone to induce oocyte fertilization. Abnormalities rely at least in part on alteration in vitamin E transfer, i.e., a new function of PLTP that has recently been shown to operate in mouse plasma (4), mouse arterial wall (4, 5, 21), mouse brain (9), and human plasma (22). In support of the latter view, vitamin E, that has longer been recognized as a key factor in spermatogenesis and reproductive biology (10, 23) is reported for the first time to be markedly reduced in spermatozoa and epididymis of PLTP-/- mice.

Although PLTP was initially recognized as a phospholipid transfer protein, which can modify HDL structure and composition (13), its biological function recently appeared to be much more complex in nature. In particular, recent identification of its role in vitamin E metabolism in vivo
has been a subject of intense research over the past few years. In direct support of a key role of PLTP in vitamin E metabolism, high vitamin E requirements were proven to be dependent in part on PLTP in several tissues (4, 6, 9). Results of the present study indicate that male reproductive organs might well be one of those. While vitamin E was discovered in 1922 as an essential determinant of mammalian fertility, in particular through its ability to maintain normal spermatogenesis (10), it is demonstrated for the first time that epididymis, in particular in its caput region is a tissue with unrivalled, elevated PLTP expression levels, exceeding sevenfold those measured in testis or liver.

Other vitamin E transport factors, including in particular scavenger receptor class B type 1 (SR-B1), α-tocopherol transfer protein (α-TTP) and ATP binding cassette transporter A1 (ABC-A1), were previously shown to influence reproductive biology through the determination of oocyte maturation, placentation, and fetal development (24–27). Implications of SR-B1, α-TTP, and ABC-A1 were shown to relate at least in part to their ability to govern vitamin E transport and metabolism. In this context, PLTP seems to provide a different, more specialized function in reproductive biology. First, it constitutes a unique vitamin E transfer factor that can affect selectively male, but not female, fertility. Second, unlike SR-B1 deficiency that produces a 77% decrease in the vitamin E content of the testis (28), neither the morphology nor the vitamin E content of testis is modified by PLTP deficiency. Third, PLTP arises as a unique vitamin E transporter that acts primarily at the well-defined, post-testicular stage of epididymal spermatozoa maturation. Fourth, whereas no fertility defect was reported in mouse males with either SR-B1, ABC-A1, or α-TTP deficiency, vitamin E deficiency in mature spermatozoa of PLTP-deficient mice results in selective decreases in spermatozoa motility and oocyte fertilization rate. Strikingly, the latter two aspects are well known to be strongly dependent on post-testicular maturation that occurs along the epididymal transit (29). Altogether, these observations indicate that observed motility defects of spermatozoa from PLTP-deficient mouse are most likely a direct consequence of vitamin E deficiency per se. It is worthy to note that vitamin E was shown in some previous studies to constitute a significant determinant of the motility of mature spermatozoa in humans as well (30). The relative proportion of motile spermatozoa in humans is strongly correlated with sperm α-tocopherol content, which may undergo considerable variation from one individual to the other (30). In addition, vitamin E supplementation in humans was reported to improve both sperm motility (31, 32) and in vitro fertilization rate (33, 34).

The two well-identified molecular properties of vitamin E, i.e., its antioxidative and membrane destabilizing properties, might be relevant in relating the PLTP-mediated, vitamin E deficiency to the reduction of spermatozoa motility and fertility. First, the decrease in sperm antioxidants, among them vitamin E, is known to favor the production of reactive oxygen species that are known to reduce spermatozoa motility and sperm-oocyte fusion (35–37). Second, and in addition to its antioxidative properties, vitamin E can influence membrane stability (38, 39), and in earlier studies, erythrocytes were induced to fuse in the presence of α-tocopherol as a result of destabilization of the membrane lipid bilayer (40). As erythrocytes, spermatozoa behave singly and they are surrounded by an aqueous phase. In this context, the membrane destabilizing effect of vitamin E might well have contributed to both flagellum motility and oocyte penetration.

In conclusion, vitamin E transfer activity of PLTP provides a new molecular mechanism, which affects male fertility. The defect associated with PLTP deficiency seems to be restricted to
spermatozoa maturation along the course of its epididymal transit, resulting in selective reductions in spermatozoa motility and oocyte fertilization. It is worthy to note that PLTP arose over the past decade as a complex factor that is expressed at significant levels in a number of tissues, including blood and brain in addition to epididymis. In this context, creation of new, tissue-specific PLTP-knocked out mouse lines will be probably of considerable help in figuring with greater details the implication of PLTP and vitamin E transfers in reproductive biology, independently of other changes. Additional studies are also needed to establish whether sperm function can be normalized in vitro and in vivo through vitamin E supplementation. Interestingly, whereas recent observations revealed that PLTP is also expressed at elevated levels in human seminal plasma (41), the relevance of vitamin E supplementation in the treatment of human male infertility has been a matter of debate over the past decade. Whether the expected benefit of vitamin E supplementation is dependent on both the epididymal expression of PLTP and the initial vitamin E content of spermatozoa in treated patients will deserve further attention.

## ACKNOWLEDGMENTS

This work was supported by INSERM, the Université de Bourgogne, the Conseil régional de Bourgogne, the Centre Hospitalier Universitaire de Dijon, and the Fondation de France.

## REFERENCES

1. Tall, A. (1995) Plasma lipid transfer proteins. *Annu. Rev. Biochem.* **64**, 235–257

2. Lagrost, L., Desrumaux, C., Masson, D., Deckert, V., and Gambert, P. (1998) Structure and function of the plasma phospholipid transfer protein. *Curr. Opin. Lipidol.* **9**, 203–209

3. Jiang, X. C., Qin, S., Qiao, C., Kawano, K., Lin, M., Skold, A., Xiao, X., and Tall, A. R. (2001) Apolipoprotein B secretion and atherosclerosis are decreased in mice with phospholipid-transfer protein deficiency. *Nat. Med.* **7**, 847–852

4. Jiang, X. C., Tall, A. R., Qin, S., Lin, M., Schneider, M., Lalanne, F., Deckert, V., Desrumaux, C., Athias, A., Witztum, J. L., et al. (2002) Phospholipid transfer protein deficiency protects circulating lipoproteins from oxidation due to the enhanced accumulation of vitamin E. *J. Biol. Chem.* **277**, 31850–31856

5. Yang, X. P., Yan, D., Qiao, C., Liu, R. J., Chen, J. G., Li, J., Schneider, M., Lagrost, L., Xiao, X., and Jiang, X. C. (2003) Increased atherosclerotic lesions in apoE mice with plasma phospholipid transfer protein overexpression. *Arterioscler. Thromb. Vasc. Biol.* **23**, 1601–1607

6. Jiang, X. C., Li, Z., Liu, R., Yang, X. P., Pan, M., Lagrost, L., Fisher, E. A., and Williams, K. J. (2005) Phospholipid transfer protein deficiency impairs apolipoprotein-B secretion from hepatocytes by stimulating a proteolytic pathway through a relative deficiency of vitamin E and an increase in intracellular oxidants. *J. Biol. Chem.* **280**, 18336–18340
7. Schlitt, A., Bickel, C., Thumma, P., Blankenberg, S., Rupprecht, H. J., Meyer, J., and Jiang, X. C. (2003) High plasma phospholipid transfer protein levels as a risk factor for coronary artery disease. *Arterioscler. Thromb. Vasc. Biol.* **23**, 1857–1862

8. Schlitt, A., Liu, J., Yan, D., Mondragon-Escorpizo, M., Norin, A. J., and Jiang, X. C. (2005) Anti-inflammatory effects of phospholipid transfer protein (PLTP) deficiency in mice. *Biochim. Biophys. Acta* **1733**, 187–191

9. Desrumaux, C., Risold, P. Y., Schroeder, H., Deckert, V., Masson, D., Athias, A., Laplanche, H., Le Guern, N., Blache, D., Jiang, X. C., et al. (2005) Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice. *FASEB J.* **19**, 296–297

10. Evans, H. M., and Bishop, K. S. (1922) On the existence of a hitherto unrecognized dietary factor essential for reproduction. *Science* **56**, 650–651

11. Albers, J. J., Wolfbauer, G., Cheung, M. C., Day, J. R., Ching, A. F. T., Lox, S., and Tu, A. Y. (1995) Functional expression of human and mouse plasma phospholipid transfer protein: effect of recombinant and plasma PLTP on HDL subspecies. *Biochim. Biophys. Acta* **1258**, 27–34

12. Ingold, K. U., Burton, G. W., Foster, D. O., Hugues, L., Lindsay, D. A., and Webb, A. (1987) Biokinetics and discrimination between dietary RRR- and SRR-alpha-tocopherols in the male rat. *Lipids* **22**, 163–172

13. Jiang, X. C., Bruce, C., Mar, J., Liu, M., Ji, Y., Francone, O. L., and Tall, A. R. (1999) Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels. *J. Clin. Invest.* **103**, 907–914

14. Vallee, M., Rivera, J. D., Koob, G. F., Purdy, R. H., and Fitzgerald, R. L. (2000) Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry. *Anal. Biochem.* **287**, 153–166

15. Wyrobek, A. J., and Bruce, W. R. (1975) Chemical induction of sperm abnormalities in mice. *Proc. Natl. Acad. Sci. USA* **72**, 4425–4429

16. Eliasson, R. (1997) Supravital staining of human spermatozoa. *Fertil. Steril.* **28**, 1257

17. World Health Organization 2000. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press, Cambridge, England

18. Katsanidis, E., and Addis, P. B. (1999) Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue. *Free Radic. Biol. Med.* **27**, 1137–1140

19. Lilford, R., Jones, A. M., Bishop, D. T., Thornton, J., and Mueller, R. (1994) Case-control study of whether subfertility in men is familial. *BMJ* **309**, 570–573
20. De Kretser, D. M., and Baker, H. W. (1999) Infertility in men: recent advances and continuing controversies. *J. Clin. Endocrinol. Metab.* **84**, 3443–3450

21. Desrumaux, C., Deckert, V., Athias, A., Masson, D., Lizard, G., Palleau, V., Gambert, P., and Lagrost, L. (1999) Plasma phospholipid transfer protein prevents vascular endothelium dysfunction by delivering alpha-tocopherol to endothelial cells. *FASEB J.* **13**, 883–892

22. Schneider, M., Verges, B., Klein, A., Miller, E. R., Deckert, V., Desrumaux, C., Masson, D., Gambert, P., Brun, J. M., Fruchart-Najib, J., et al. (2004) Alterations in plasma vitamin E distribution in type 2 diabetic patients with elevated plasma phospholipid transfer protein activity. *Diabetes* **53**, 2633–2639

23. Kaempf-Rotzoll, D. E., Traber, M. G., and Arai, H. (2003) Vitamin E and transfer proteins. *Curr. Opin. Lipidol.* **14**, 249–254

24. Trigatti, B., Rayburn, H., Vinals, M., Braun, A., Miettinen, H., Penman, M., Hertz, M., Schrenzel, M., Amigo, L., Rigotti, A., et al. (1999) Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology. *Proc. Natl. Acad. Sci. USA* **96**, 9322–9327

25. Miettinen, H. E., Rayburn, H., and Krieger, M. (2001) Abnormal lipoprotein metabolism and reversible female infertility in HDL receptor (SR-BI)-deficient mice. *J. Clin. Invest.* **108**, 1717–1722

26. Jishage, K., Arita, M., Igarashi, K., Iwata, T., Watanabe, M., Ogawa, M., Kamada, N., Inoue, K., Arai, H., and Suzuki, H. (2001) Alpha-tocopherol transfer protein is important for the normal development of placental labyrinthine trophoblasts in mice. *J. Biol. Chem.* **276**, 1669–1672

27. Christiansen-Weber, T. A., Voland, J. R., Wu, Y., Ngo, K., Roland, B. L., Nguyen, S., Peterson, P. A., and Fung-Leung, W. P. (2000) Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency. *Am. J. Pathol.* **157**, 1017–1029

28. Mardones, P., Strobel, P., Miranda, S., Leighton, F., Quitones, V., Amigo, L., Rozowski, J., Krieger, M., and Rigotti, A. (2002) Alpha-tocopherol metabolism is abnormal in scavenger receptor class B type I (SR-BI)-deficient mice. *J. Nutr.* **132**, 443–449

29. Gatti, J. L., Castella, S., Dacheux, F., Ecroyd, H., Metayer, S., Thimon, V., and Dacheux, J. L. (2004) Post-testicular sperm environment and fertility. *Anim. Reprod. Sci.* **82-83**, 321–339

30. Therond, P., Auger, J., Legrand, A., and Jouannet, P. (1996) Alpha-Tocopherol in human spermatozoa and seminal plasma: relationships with motility, antioxidant enzymes and leukocytes. *Mol. Hum. Reprod.* **2**, 739–744
31. Suleiman, S. A., Ali, M. E., Zaki, Z. M., el-Malik, E. M., and Nasr, M. A. (1996) Lipid peroxidation and human sperm motility: protective role of vitamin E. *J. Androl.* **17**, 530–537

32. Keskes-Ammar, L., Feki-Chakroun, N., Rebai, T., Shanoun, Z., Ghozzi, H., Hammami, S., Zghal, K., Fki, H., Damak, J., and Bahloul, A. (2003) Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men. *Arch. Androl.* **49**, 83–94

33. Kessopoulou, E., Powers, H. J., Sharma, K. K., Pearson, M. J., Russel, J. M., Cooke, I. D., and Barratt, C. L. (1995) A double-blind randomized placebo cross-over controlled trial using the antioxidant vitamin E to treat reactive oxygen species associated male infertility. *Fertil. Steril.* **64**, 825–831

34. Geva, E., Bartoov, B., Zabludovsky, N., Lessing, J. B., Lerner-Geva, L., and Amit, A. (1996) The effect of antioxidant treatment on human spermatozoa and fertilization rate in an *in vitro* fertilization program. *Fertil. Steril.* **66**, 430–434

35. Agarwal, A., Saleh, R.A., and Bedaiwy, M.A. (2003) Role of reactive oxygen species in the pathophysiology of human reproduction. *Fertil. Steril.* **79**, 829–843

36. Agarwal, A., Allamaneni, S.S., Nallella, K.P., and Mascha, E. (2005) Correlation of reactive oxygen species levels with the fertilization rate after in vitro fertilization: a qualified meta-analysis. *Fertil. Steril.* **84**, 228–231

37. Breininger, E., Beorlegui, N.B., O'Flaherty, C.M., and Beconi, M.T. (2005) Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen. *Theriogenology.* **63**, 2126–2135

38. Neild D. M., Brouwers, J.F., Colenbrander, B., Aguero, A., and Gadella, B.M. (2005) Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa. *Mol Reprod Dev.* **72**, 230–8

39. Pena, F.J., Johannisson, A., Wallgren, M., and Rodriguez Martinez, H. (2004) Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture. *Zygote* **12**, 117–124

40. Ahkong, Q.F., Fisher, D., Tampion, W., Lucy, J.A. (1973) The fusion of erythrocytes by fatty acids, esters, retinol and α-tocopherol. *Biochem. J.* **136**, 147–155

41. Masson, D., Drouineaud, V., Moiroux, P., Gautier, T., Dautin, G., Schneider, M., Fruchart-Najib, J., Jauhiainen; M., Ehnholm; C., Sagot; P., Gambert; P., Jimenez, C., and Lagrost, L. (2003) Human seminal plasma displays significant phospholipid transfer activity due to the presence of active phospholipids transfer protein. *Molecular Human Reproduction* **9**, 457–464

Received November 14, 2005; accepted December 22, 2005.
<caption>**Table 1**</caption>

Alpha-tocopherol content of spermatozoa, testis and epididymis.

<table><thead><tr><th rowspan="3"></th><th colspan="5">Alpha-tocopherol</th></tr><tr><th colspan="2">Spermatozoa<br>(ng/10<sup>6</sup> cells)<br>(n=9)</th><th>Testis (ng/mg)<br>(n=9)</th><th colspan="2">Epididymis<br>(ng/mg)<br>(n=3)</th></tr><tr><th>From caput<br>epididymis</th><th>From cauda<br>epididymis</th><th>Caput</th><th colspan="2">Cauda</th></tr></thead><tbody><tr><td>WT</td><td>13.8 ± 2.4</td><td>3.1 ± 0.9</td><td>7.4 ± 0.9</td><td>22.6 ± 2.7</td><td>13.0 ± 0.5</td></tr><tr><td>PLTP-/-</td><td>9.8 ± 1.7</td><td>2.3 ± 0.8</td><td>6.7 ± 0.6</td><td>16.8 ± 0.5</td><td>10.3 ± 0.2</td></tr><tr><td>P</td><td>&lt;0.05</td><td>&lt;0.03</td><td>NS</td><td>&lt;0.05</td><td>&lt;0.05</td></tr></tbody></table>

Tissues were homogenized, and alpha-tocopherol was extracted and quantitated by GC-MS as described in Materials and Methods. Values are mean ± SEM. For spermatozoa and testis analyses, paired samples of one WT mouse male and one PLTP-/- mouse male with the same age were analyzed simultaneously. Statistical significance of differences was calculated by the Wilcoxon signed rank test. Epididymal values were obtained in an independent set of experiments. Analysis was conducted with WT and PLTP-/- mouse males of the same age (25 week-old) and sacrificed on the same day. In this case, statistical significance was calculated by Mann-Whitney test.
**Table 2**

Weights of reproductive organs in WT and PLTP-/- mice

<table><thead><tr><th rowspan="2"></th><th colspan="6">Organ weight (g per 100 g body weight)</th></tr><tr><th>Body (n=18)</th><th>Testis (n=18)</th><th>Epididymis (n=12)</th><th>Prostatis (n=12)</th><th>Vesicular glands (n=12)</th><th>Coagulating glands (n=12)</th></tr></thead><tbody><tr><td>WT</td><td>33.6 ± 1.6</td><td>0.64 ± 0.02</td><td>0.23 ± 0.01</td><td>0.12 ± 0.03</td><td>1.25 ± 0.17</td><td>0.12 ± 0.01</td></tr><tr><td>PLTP-/-</td><td>33.6 ± 1.5</td><td>0.58 ± 0.02</td><td>0.23 ± 0.01</td><td>0.12 ± 0.01</td><td>1.16 ± 0.24</td><td>0.11 ± 0.01</td></tr><tr><td>P</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr></tbody></table>

Ratio of organ to body weight was calculated for 12-66 week-old WT and PLTP-/- mouse males. Values are mean ± SEM. Statistical analysis by Wilcoxon signed rank test.
**Table 3**

Characteristics of spermatozoa from cauda epididymis.

<table><thead><tr><th rowspan="2"></th><th colspan="3">Spermatozoa characteristics</th></tr><tr><th>Count<br>(×10<sup>6</sup>)</th><th>Viability (%)</th><th>Morphology<br>(% normal)</th></tr></thead><tbody><tr><td>WT (n=13)</td><td>29 ± 5</td><td>60 ± 2</td><td>57 ± 2</td></tr><tr><td>PLTP-/- (n=13)</td><td>23 ± 3</td><td>57 ± 3</td><td>52 ± 2</td></tr></tbody></table>

Spermatozoa parameters of 12–33 week-old WT and PLTP-/- mice were determined as described in Materials and Methods. Values are mean ± SEM. Paired samples of one WT mouse male and one PLTP-/- mouse male with the same age were analyzed simultaneously. Statistical significance of differences was calculated by the Wilcoxon signed rank test.
<figure><img src="image_4.png" /><figcaption>Fig. 1</figcaption></figure>

**Figure 1.** Effect of PLTP deficiency on male and female fertility. Total number of pups was examined in a continuous breeding study over a 2 month period. Four categories of breeders were formed: Wild-type (WT) male with WT female mice (WT×WT; *n*=16), PLTP-/- male with PLTP-/- female mice (PLTP-/-×PLTP-/-; *n*=18), PLTP-/- male with WT female (WT×PLTP-/- (male), *n*=7) and, WT male with PLTP-/- female (WT×PLTP-/- (female); *n*=5). Statistical significance was calculated by the non-parametric Mann-Whitney test.
<figure><img src="image_5.png" /><figcaption>Fig. 2</figcaption></figure>

**Figure 2.** Quantitative PCR analysis of PLTP expression in various organs of WT mice. The expression levels of PLTP in testis, caput and cauda epididymis, prostatis, vesicular glands, coagulating glands and liver of WT mice were determined by quantitative reverse transcription-PCR as described in Materials and Methods. Data are standardized for 18S RNA. Values are mean ± SEM of 17-week-old wild-type mice (n=4).
<figure><img src="image_6.png" /><figcaption>Fig. 3</figcaption></figure>

**Figure 3.** Immunolocalization of PLTP in testis, caput and cauda epididymis from PLTP-/- and WT mice. Immunostaining was performed using a polyclonal PLTP specific antiserum (1/100) on testis (A), caput (B), and cauda (C) epididymis sections from PLTP-/- mice (**left panels**) and wild-type mice (**middle panels**). In **right panels**, the same histological sections corresponding to WT testis or epididymis regions were stained with hematoxylin-eosin. Original magnification, 200×. Ep, epithelium; Lu, lumen; ST, seminiferous tubule, spz, spermatozoa.
<figure><img src="image_7.png" /><figcaption>Fig. 4</figcaption></figure>

**Figure 4.** Structural organization of seminiferous tubules of WT and PLTP-/- mice. Testis were obtained from 21 to 23 week-old WT and PLTP-/- mouse males, paraffin-embedded, and sections were stained with hematoxylin-eosin. Representative sections are shown. Lu, lumen; ST, seminiferous tubule; L, Leidig cell; spg, spermatogonia; spc, spermatocyte; r spt, round spermatid; e spt, elongated spermatid, spz, spermatozoa. Magnification 200× (**left panels**) and 400× (**insert** and **right panels**).
<figure><img src="image_8.png" /><figcaption>Fig. 6</figcaption></figure>

**Figure 6.** Effect of PLTP deficiency on in vitro fertilization rate. In vitro fertilization was assessed by incubating oocytes from wild-type females in the presence of spermatozoa from either wild-type (**left**) or PLTP-/- (**right**) mouse males; 37 to 38 oocytes were used per spermatozoa suspensions which were obtained from WT (n=5) and PLTP-/- (n=5) mouse epididymis. The appearance of two-cell-stage embryos was taken to indicate successful penetration of sperm and subsequent activation of fertilization. Original magnification 400×. Statistical significance was calculated by the non-parametric Wilcoxon signed rank test.