No obvious phenotypic abnormalities in mice lacking the Pate4 gene

Highlights

• Pate4 expression in bone-resorbing osteoclasts is negatively regulated by calcitonin.

• Pate4-deficient mice do not display a bone remodeling phenotype.

• Pate4 is predominantly expressed in prostate and seminal vesicles.

• Pate4-deficiency does not reduce fertility.

Abstract

We have previously reported that the hormone calcitonin (CT) negatively regulates bone formation by inhibiting the release of sphingosine-1-phosphate from bone-resorbing osteoclasts. In the context of this study we additionally observed that CT repressed the expression of Pate4, encoding the secreted protein caltrin/Svs7, in osteoclasts from wildtype mice. To assess a possible function of Pate4 in bone remodeling, we utilized commercially available embryonic stem cells with a targeted Pate4 allele to generate Pate4-deficient mice. These were born at the expected Mendelian ratio and did not display obvious abnormalities until the age of 6 months. A bone-specific histomorphometric analysis further revealed that bone remodeling is unaffected in male and female Pate4-deficient mice. Since a subsequently performed multi-tissue expression analysis confirmed that Pate4 is primarily expressed in prostate and seminal vesicles, we additionally analyzed the respective tissues of Pate4-deficient mice, but failed to detect histological abnormalities. Most importantly, as assessed by mating with female wildtype mice, we did not observe reduced fertility associated with Pate4-deficiency. Taken together, our study was the first to generate and analyze a mouse model lacking Pate4, a gene with strong expression in prostate and seminal vesicles, yet without major function for fertility.

Introduction

The PATE (prostate and testis expressed) locus on human chromosome 11q24.2 is comprised of five genes, now designated ACRV1 and PATE1-4, encoding secreted proteins with similar domain structure [1]. Whereas the founding member, PATE1, was initially identified as a gene with selective expression in prostate and testis [2], the other PATE genes were found to be expressed with similar specificity, although the abundance of mRNA transcripts varied between the two tissues. More specifically, while PATE2 and PATE3 were found primarily expressed in testis, PATE4 expression was more pronounced in prostate [1]. At the protein level, homologues of human PATE4, were originally identified by purification from rat or mouse seminal vesicle content and termed caltrin or Svs7 (thereafter generally termed Pate4) [3], [4], [5]. At a functional level it was shown caltrin/Pate4 inhibits calcium uptake by rat spermatozoa [3] and reduces acrosomal exocytosis of sperm cells thereby increasing sperm–egg interaction [4]. It was further reported that Svs7/Pate4 enhances sperm motility in vitro via binding to phospholipids of the sperm surface [5]. Although these collective findings suggested an involvement of Pate4 in male fertility, their physiological relevance has not been addressed so far in vivo.

Our reasoning to generate and analyze Pate4-deficient mice came from a genome-wide expression analysis performed in the context of a study on the role of calcitonin (CT) in bone remodeling [6]. More specifically, CT is a thyroid-derived hormone blocking osteoclast-mediated bone resorption, when administered at pharmological doses [7], [8], [9]. Physiologically however, CT primarily acts as an inhibitor of osteoblast-mediated bone formation, which was first identified through the analysis of CT-deficient mice displaying a high bone mass phenotype [10], [11], [12]. Since the CT receptor (CTR) however is strongly expressed by osteoclasts, not by osteoblasts, it appeared that the function of CT in bone remodeling is mediated indirectly, i.e. through regulation of bone remodeling coupling factors released by osteoclasts and acting on osteoblasts [13], [14], [15]. To confirm this hypothesis we utilized Cre-LoxP technology to demonstrate a high bone mass phenotype in mice with specific CTR inactivation in osteoclasts [6]. In an attempt to identify CT-regulated genes we treated primary osteoclasts from wildtype and CTR-deficient mice with CT and performed genome-wide expression analysis, thereby identifying 29 genes, whose transcription was regulated by CT specifically in wildtype cultures [6]. While one of the genes encoded the sphingosine-1-phosphate transporter Spns2 [16], [17], whose transcriptional repression by CT potentially explained the physiological role of CT in bone remodeling, the other differentially expressed genes did not offer an immediate explanation for the phenotypes of CT and CTR-deficient mice.

One of these CT-regulated genes was Pate4, which encodes a secreted protein with unknown physiological function. Here we show, by generating a Pate4-deficient mouse model, that Pate4-deficiency does not affect bone remodeling, consistent with the fact that it is predominantly expressed in prostate and seminal vesicles. Importantly however, the absence of Pate4 from both organs did not cause obvious phenotypic abnormalities, and Pate4-deficient male mice did not display decreased fertility. Taken together, our data provide the first description of a Pate4-deficient mouse model, which does not display specific organ defects despite the unique expression pattern of Pate4.