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Phospholipid transfer protein deficiency reduces sperm motility and impairs fertility of mouse males

Véronique Drouineaud^*, Laurent Lagrost^,,1, Alexis Klein^,, Catherine Desrumaux^,, Naig Le Guern^,, Anne Athias^,, Franck Ménétrier, Philippe Moiroux^*, Paul Sagot, Clément Jimenez^*, David Masson^, and Valérie Deckert^,,1

^* Laboratoire de Biologie de la Reproduction, EA Génétique et Reproduction 3185, CHU Dijon, 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

¹Correspondence: INSERM U498, Faculté de Médecine, 7 Bd Jeanne d’arc, BP87900, Dijon 21079, France. E-mail: laurent.lagrost{at}u-bourgogne.fr; valerie.deckert{at}u-bourgogne.fr

SPECIFIC AIMS

Vitamin E’s implication in reproductive biology has long been known, and recently its transport in mammalian plasma and brain has been shown to be governed by plasma phospholipid transfer protein (PLTP). The effect of PLTP deficiency on mouse fertility was addressed in the present study.

PRINCIPAL FINDINGS

1. PLTP deficiency affects male, but not female fertility
To characterize the effect of PLTP deficiency on reproductive function, a continuous breeding study was conducted in wild-type (WT) and PLTP–/– mice over a 2 month period. The number of days to the first litter did not differ between PLTP–/– and WT 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 (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 WT counterparts (–32%; P<0.03). 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 WT 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.

2. Epididymis is one of the strongest PLTP expressors among tissues of mouse males
A widespread distribution of PLTP was found 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. The highest PLTP staining intensity was preferentially localized in the apical surface of epididymal principal cells in the caput region (not shown).

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 (not shown). Beyond the morphological analysis, epididymis and spermatozoa were analyzed for their -tocopherol content, which is known to constitute a key factor in reproductive biology. As shown in Table 1 , -tocopherol concentration was significantly lower in cauda and caput epididymis of PLTP–/– mice as compared with WT 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 -tocopherol content of epididymis (Table 1) ; and PLTP deficiency induced a significant decrease in the -tocopherol content of spermatozoa from PLTP–/–mice as compared with spermatozoa from WT mice (caput: –29%, P<0.01; cauda: –26%, P<0.05).

4. 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 (not shown). No significant difference in plasma and testicular testosterone levels were observed between WT 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). 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 WT mice (not shown).

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 WT females were incubated for 24 h with capacitated WT or PLTP–/– spermatozoa originated from the epididymis cauda. The appearance of two-cell-stage embryos was taken to indicate successful penetration of spermatozoa and subsequent activation of fertilization. As shown in Fig. 1 , 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).

View larger version (67K): [in this window] [in a new window]   Figure 1. Effect of PLTP deficiency on in vitro fertilization rate. In vitro fertilization was assessed by incubating oocytes from WT females in the presence of spermatozoa from either WT (left) or PLTP–/– (right) mouse males. 37 to 38 oocytes were used per spermatozoa suspension, 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 spermatozoa and subsequent activation of fertilization. Original magnification x400. Statistical significance was calculated by the nonparametric Wilcoxon signed rank test.

CONCLUSIONS AND SIGNIFICANCE

In the present study, plasma phospholipid transfer protein (PLTP) (i.e., a protein normally expressed at high levels in several mouse and human tissues) arose as a new candidate that may play a crucial role in reproductive biology. PLTP deficiency is associated with a lower fertility of mouse males, but not mousefemales. Spermatozoa motility is significantly decreased in PLTP–/– mouse males, and 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 was recently shown to operate in mouse plasma, mouse arterial wall, mouse brain, and human plasma). In support of the latter view, vitamin E, long recognized as a key factor in spermatogenesis and reproductive biology, is reported for the first time to be markedly reduced in spermatozoa and epididymis of PLTP–/– mice. Compared with other previously identified vitamin E transport factors, PLTP seems to provide a different, more specialized function in reproductive biology. First, it constitutes a unique vitamin E transfer factor that is able to selectively affect male, but not female fertility. Second, 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, 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. Altogether, these observations indicate that observed motility defects of spermatozoa from PLTP-deficient mice is most likely a direct consequence of vitamin E deficiency per se. Whereas the relevance of vitamin E supplementation in the treatment of human male infertility has been a matter of debate over the last decade, the present study suggests that the expected benefit of vitamin E supplementation might well be dependent on both the epididymal expression of PLTP and the initial vitamin E content of spermatozoa in treated patients. The latter hypothesis will deserve further attention.

View larger version (19K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5385fje;

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