Impaired sperm maturation in conditional Lcn6 knockout mice

Impaired sperm maturation in conditional Lcn6 knockout mice Abstract Human LCN6, a lipocalin protein, exhibits predominant expression in epididymis and location on the sperm surface. However, the biological function of LCN6 in vivo remains unknown. Herein, we found that unlike human LCN6, mouse Lcn6 gene encoded two transcript variants that were both upregulated by androgen. Subsequently, we generated a conditional knockout mouse model to disrupt Lcn6 in the adult and investigate its function. In this model, spermatogenesis was normal and Lcn6 deficiency did not affect the natural birth rate of male mice or in vitro fertilization ability of their cauda epididymal sperm. Nevertheless, sperm from the cauda epididymis of the Lcn6 null mice underwent a sustained increase of acrosome reaction frequency whether capacitated or not (P < 0.01). Consistent with premature acrosome reaction, sperm from knockout mice had significantly increased intracellular calcium content when extracellular calcium was supplied (P < 0.01). These results demonstrate an important function of LCN6 in preventing calcium overload and premature acrosome reaction of sperm and suggest a potential risk factor of LCN6 deficiency for sperm maturation. Introduction Sperm accomplishing the developmental stage in the testis are immotile and immature. They gain progressive motility and undergo maturation during their transit through epididymis. This maturation process is thought to be completed in the interaction between sperm and proteins synthesized and secreted by the epididymal epithelium [1–3]. The mammalian epididymis is divided into three regions: caput, corpus, and cauda. In fact, the most proximal caput region of rodents’ epididymides exhibits a feature distinct from the other caput region, thus, this special part is named the initial segment. Each segment of epididymis has its unique gene expression patterns, leading to that diverse secretory proteins function in sperm maturation sequentially [4–6]. But the specific roles of these epididymal proteins, especially that exclusively or predominantly expressed in epididymis, are largely unknown. Lipocalin family is an ancient superfamily of extracellular proteins that function in a broad range of systems including taste and odor chemoreception and transport, coloration, immune modulation, prostaglandin D synthesis, and metabolism [7–12]. Its involvement in sperm maturation was highlighted by several reports. LCN2, also known as 24p3, was reported to act as a suppressor of acrosome reaction (AR) [13], and to enhance sperm motility through elevation of intracellular pH and increase of intracellular cyclic adenosine monophosphate accumulation [14]. LCN5, the epididymal retinoic acid binding protein, was indicated to function in the maintenance of epithelium of cauda epididymis [15]. However, the understanding about lipocalins on male fertility is far from complete. In mouse and human, there is a highly conserved lipocalin gene cluster with similar gene number, order, and orientation. Within the cluster, Lcn5, Lcn8, Lcn9, Lcn10, Lcn12, and Lcn13 were first cloned and proved specifically expressed in the mouse epididymis [16]. Previously, we cloned a novel lipocalin gene named Lcn6 that is adjacent to Lcn5 and Lcn8 (see Supplementary Figure S1). The encoded protein in human was predominantly located in epididymis and associated with the sperm head and neck [17], suggesting the importance of LCN6 in male fertility. Conventional knockout mouse models have been widely used in the functional research for epididymal proteins. For example, cSrc, transaldolase, and c-ros receptor tyrosine kinase were demonstrated their importance in epididymal development and sperm maturation [18–20]. But it is noteworthy that a number of mice with deletion of epididymis-specific genes generated by conventional knockout approaches do not show fertility failure phenotype [21–23]. This phenomenon can be attributed to some developmental compensatory mechanism that is common in other organs and species and thought to be created during the evolution [24–26]. In addition, a recent work describing that homozygous deletion of a cluster of nine β-defensin genes in the mouse epididymis results in male sterility sincerely supports the existence of compensation [27]. For this reason, establishment of conditional gene null mouse models to elucidate the epididymal function is required. Nevertheless, there are scarce research papers using conditional knockout mouse model to research on epididymis [28,29], and induced gene disruption in adult epididymis has not been reported. Here, we established a knockout mouse model in which Lcn6 deletion was achieved in the adult mice by tamoxifen (TM) administration to investigate the in vivo function of LCN6 on male fertility. Materials and methods Animals, tissue preparation All the experimental procedures were carried out according to the protocol with the approval of the Institutional Animal Care and Use Committee (approved number: SIBCB-S281-1510-2-041). Mice of the wild-type (WT) strain C57BL/6 were supplied by the Animal Center of the Chinese Academy of Sciences (Shanghai, China). Prior to dissection, animals were euthanized with CO2 inhalation. Tissues for mRNA analysis were excised and frozen immediately in liquid nitrogen, tissues for in situ hybridization and Hematoxylin and Eosin (HE) staining were fixed in 4% paraformaldehyde (PFA) or Bouin fluid for further process. Castration and androgen replacement 8-week-old WT male mice were castrated bilaterally under sodium pentobarbital anesthesia. Animals were divided into 9 groups (3–6 mice per group), and killed on Day 0, 1, 3, 5, 7 after castration as well as 1, 3, 5, 7 days after the initial testosterone propionate injection. Androgen supplementation began on the seventh day after castration at the dose of 4 mg/kg body weight. Changes in the serum testosterone levels of each group were confirmed by radioimmunoassay in Shanghai Zhongshan Hospital. Total RNA isolation and Northern blot analysis Total RNA was extracted from tissue homogenates with TRIzol reagents (Invitrogen, USA) according to the manufacturer's instructions. Northern blot analysis was performed as described previously [30]. Briefly, 20 μg of total RNA from each sample was subjected to 1.2% (w/v) agarose-formaldehyde gel electrophoresis, blotted onto nylon membranes by capillary transfer, and hybridized with a probe that was a 32P-labeled cDNA fragment of mouse Lcn6. The hybridization signal from 18S rRNA was used as loading control. Autoradiographs with pronounced differences in expression were analyzed by densitometry. The probes are amplified from epididymal cDNA using the primers listed in Supplementary Table S1. Reverse transcription polymerase chain reaction (RT-PCR) and Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) The first strand of cDNA was synthesized with a reverse transcription (RT) kit (Takara, FSQ-301) from mRNA extract. Then the products were diluted 25-fold and subjected to PCR using reagents of 2× Taq PCR Master Mix from Takara (Japan). qRT-PCR was performed with 2× SYBR Green qPCR Master Mix from Bimake (USA) on an ABI QuantStudio 6 Flex Real-Time PCR Detection System (Applied Biosystems, USA). All the primers are listed in Supplementary Table S1. In situ hybridization In situ hybridization of Lcn6 mRNA in WT epididymis was performed following the protocol previously described [31] with a few modifications. Firstly, a fragment of corresponding Lcn6α cDNA was correctly cloned into the pGEMT-Easy Vector (Promega, A1360). Secondly, the digoxigenin-labeled probe RNA was synthesized through in vitro transcription with SP6 and T7 RNA polymerase using DIG RNA labeling Kit (Roche, 1175025). Thirdly, after incubation of epididymal sections and digoxigenin-labeled probe, alkaline phosphatase coupled with anti-digoxigenin antibody (Roche, 11093274910) was used and the in situ signal was developed with NBT/BCIP system (Roche, 11681451001). The sense probe was used as negative control. Primers that were used to amplify Lcn6α are listed in Supplementary Table S1. Generation of conditional Lcn6 knockout mice The 4.4 kb upstream and 3.5 kb downstream homologous arms flanking exon 2 were amplified by PCR from 129/Sv mouse genomic DNA. These fragments were cloned into the vector that had a backbone for the targeting replacement. We used PBR322 with the neomycin resistance gene (Neo) flanked by flippase recognition target (FRT) sites and loxP sites and unique restriction sites to introduce the targeted exon and the homologous arms. The final vector was electroporated into embryonic stem cells, and correctly targeted clones were screened by PCR for homologous recombination. Then chimeric mice with the genotype Lcn6fl/+; neo/+ were generated and crossed to a Flp line to obtain Neo resistance gene deletion mice carrying the Lcn6 conditional alleles (Lcn6fl/+). Subsequently, Lcn6fl/+ mice were crossed to mice expressing a TM-inducible Cre-recombinase, which is under control of the Ubiquitin C promoter, referred to as UbC-Cre/ERT2. The offsprings with the genotype Lcn6fl/+; UbC-Cre/ERT2 were backcrossed to Lcn6fl/+ to derive Lcn6fl/fl; UbC-Cre/ERT2 mice and Lcn6fl/fl mice. The Lcn6fl/fl; UbC-Cre/ERT2 offsprings obtained from the parental Lcn6fl/fl; UbC-Cre/ERT2 male mice by Lcn6fl/fl cross were for further experiments, while male Lcn6fl/fl littermates were served as controls. Cre-ERT2 recombinase was activated in adult mice at 8–12 week of age by administering TM dissolved in Corn Oil (Sigma). Mice were injected i.p. every other day for 5 days at a concentration of 2 mg/mouse (∼77 mg/kg). The day of the first injection was marked as day 0, two weeks after TM administration, TM-treated males were, respectively, caged with WT female mice at the ratio of 1:2 for two rounds (one week for a round) to evacuate sperm that already existed. Then further analyses were applied. The primers used for genotyping are listed in Supplementary Table S1. Hematoxylin and Eosin staining Fixed testes and epididymides were dehydrated using graded ethanol (0, 30%, 50%, 75%, 85%, 95%, and 100%), vitrified by dimethylbenzene and embedded in the paraffin. Then the paraffin embedding epididymides were cut into sections with thickness of 5 μM. Finally, HE staining was performed by standard procedures. Fertility assay Eligible TM-treated males were mated with 6-week-old females at the ratio of 1:2 for 6 months. During this period, the size and sex ratio of every litter were recorded. Assessment of cauda epididymal sperm motility and tyrosine phosphorylation All the procedures were performed as reported previously [32] with minor modifications. In general, the cauda epididymal sperm were released into “Biggers, Whitten, and Whittingham” (BWW) medium [33] at an appropriate concentration of 5–10×106 cells/ml. This time-point was defined as the beginning of capacitation (0 min), then the sperm were capacitated for various time periods. During the whole capacitation process (120 min), the sperm motility was assessed using a computer-assisted semen analysis machine, the sperm extracts were collected and subjected to Western blot for tyrosine phosphorylation analysis. Western blot analysis Proteins were extracted from sperm by directly mixing with 1× laemmli loading buffer (Sigma) and boiled for 10 min at 100°C. Then they were resolved by SDS/polyacrylamide gel electrophoresis using a 4% stacking gel and a 12% separating gel and transferred to a PVDF membrane (Amersham/GE). After blocking overnight, the membranes were incubated with the primary antibodies and secondary antibodies listed below to detect the protein expression. The following antibodies were used: anti-phosphotyrosine monoclonal antibody, clone 4G10 (Merck/millipore, 1:10 000), anti-α-tubulin monoclonal antibody (Sigma, 1:20 000), Goat anti-mouse IgG, H&L Chain Specific Peroxidase Conjugate (Merck/millipore, 1:10 000). In vitro fertilization In vitro fertilization was performed as described elsewhere [34] with some modifications. Briefly, 3-week-old WT female mice were superovulated and cumulus-intact oocytes were collected. Then oocytes were pooled and divided into several groups. The sperm from TM-treated males were added to the fertilization droplet containing the eggs. After 3 h or 6 h incubation, the eggs were washed to remove unbound sperm and transferred to new fertilization droplets. Twenty-four hours later, fertilization rates were evaluated by recording the number of two-cell embryo. Evaluation of sperm acrosome reaction frequency The sperm AR frequency was assessed as described elsewhere [35]. In Brief, released sperm were capacitated for the indicated time, centrifuged and washed with PBS, then spreaded onto slides, airdried and fixed with 4% PFA. Then 3 μM final concentration of fluorescein isothiocyanate conjugated lectin from Arachis hypogaea (peanut) (Sigma) was used to stain the acrosome of spermatozoa, and DNA was counterstained with 4΄,6-diamidino-2-phenylindole (Sigma). To assess the Ca2+ ionophore A23187 induced AR frequency, released sperm were capacitated for 1 h and treated with 10 μM A23187 (Sigma) for 15 min, then the above procedures were performed. Measurement of [Ca2+]i concentration The measurement of sperm intracellular calcium level was carried out as described elsewhere [36]. Spermatozoa were allowed to disperse into the BWW medium without Ca2+ or complete BWW as indicated and loaded with the acetoxy-methyl ester of fura-2 (Fura-2/AM; Sigma; 3 μM) according to the manufacturer's protocol. The dynamic range for Ca2+-dependent fluorescence signals was obtained on a BioTek Synergy NEO multifunctional microplate detector by using excitation at 340 nm and 380 nm and ratioing the fluorescence in tensities detected at ∼510 nm. The [Ca2+] i was calculated using the equation $$\,\mathop {[ {{\rm{C}}{{\rm{a}}^{{\rm{2 + }}}}} ]}\nolimits_i = {K_d}{{( {F - {F_{\min }}} )} / {( {{F_{\max }} - F} )}}$$, where Kd = 224 nM, $$F = {{{F_{340}}} / {{F_{380}}}}$$. Fmax and Fmin were recorded at the end of the incubation period after supplementation of calcium when extracellular calcium was depleted. Fmax was determined after the addition of 20 mM digitonin, and Fmin was determined after addition of 10 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N΄,N΄-tetraacetic acid, of which the pH was adjusted to 8.4 with Tris base. Each sample was measured in triplicate. Images acquisition and processing All the slides were observed and photographed under an Olympus BX51 fluorescent microscope equipped with a DP70 camera. Images to evaluate the AR frequency were processed by Image-Pro Plus (version 6.0) and then determined by manual count. At least 200 sperm were scored in each sample. Statistical analysis All of the Western blot and Northern blot images and RT-PCR results are representative of at least three independent experiments. The qRT-PCR was performed in triplicate. Data were analyzed by Prism software (version 6.01) and P < 0.05 was defined as statistically significant. Results Epididymis-specific and androgen-regulated expression of the mouse Lcn6 The mouse ortholog of human LCN6 was obtained by homology search of the mouse expressed sequence tag database at http://www.ncbi.nlm.nih.gov/BLAST based on human and rat Lcn6 cDNA sequence. The full-length cDNA of mouse Lcn6 was amplified by RT-PCR and sequenced. Unlike human LCN6, mouse Lcn6 gene encodes two transcripts named after mLcn6α and mLcn6β, respectively (Figure 1A). Both transcripts have 7 exons and 6 introns characteristic of lipocalin gene family, and they share the exact same sequence ahead of exon6. But due to alternative splicing, variant mLcn6β skips the stop codon that mLcn6α has and encodes a longer isoform than mLcn6α (Figure 1A, top panel; see Supplementary Figure S2). As predicted with SignalP 4.1 server at http://www.cbs.dtu.dk/services/SignalP, the N-terminal 21 amino acid of Lcn6 encoded proteins probably formed a signal peptide (Figure 1A, bottom panel; see Supplementary Figure S2). In the male mice, both transcripts were exclusively detected in the epididymis rather than other reproductive organs such as testis, seminal vesicle, prostate and vas deferens and other male organs (Figure 1B, left panel). While in the female, neither of them was detected in any tested tissues (Figure 1C). A further examination by Northern blot hybridization showed the specific expression of Lcn6 in the caput but not in the other portions of epididymis (Figure 1B, right panel). Moreover, using RNA in situ hybridization we confirmed the localization of Lcn6 transcripts was mainly in the proximal caput epididymis (Figure 1D). Figure 1. View largeDownload slide Schematic diagram of mLcn6 transcript variants and expression pattern of mLcn6 in WT mouse. (A) Structure of pre-mRNAs (top panel) and encoded proteins (bottom panel). Top panel: box, exon; line, intron; number indicates the length of exon or intron. Bottom panel: box, amino acid sequence; number indicates where it starts and ends. (B) Left panel: Northern blot analysis of the mLcn6 expression in 16 tissues of male mice and rat epididymis. Right panel: Northern blot analysis of mLcn6 expression in precise segments of mouse epididymis. Template from testis was used as negative control. 18S rRNA was used as loading control. (C) RT-PCR analysis of mLcn6 expression in 9 tissues from female mice. Template from mouse epididymis and template without cDNA were used as positive control and negative control (N. C.), respectively. Gapdh mRNA was used as loading control. (D) In situ hybridization analysis of Lcn6 mRNA localization (blue) in the caput epididymis. Left panel shows the full picture, right panel shows the magnified picture of the framed region of epididymis. Sense probe was used as negative control. Scale bar (left) = 500 μm, scale bar (right) = 50 μm. Figure 1. View largeDownload slide Schematic diagram of mLcn6 transcript variants and expression pattern of mLcn6 in WT mouse. (A) Structure of pre-mRNAs (top panel) and encoded proteins (bottom panel). Top panel: box, exon; line, intron; number indicates the length of exon or intron. Bottom panel: box, amino acid sequence; number indicates where it starts and ends. (B) Left panel: Northern blot analysis of the mLcn6 expression in 16 tissues of male mice and rat epididymis. Right panel: Northern blot analysis of mLcn6 expression in precise segments of mouse epididymis. Template from testis was used as negative control. 18S rRNA was used as loading control. (C) RT-PCR analysis of mLcn6 expression in 9 tissues from female mice. Template from mouse epididymis and template without cDNA were used as positive control and negative control (N. C.), respectively. Gapdh mRNA was used as loading control. (D) In situ hybridization analysis of Lcn6 mRNA localization (blue) in the caput epididymis. Left panel shows the full picture, right panel shows the magnified picture of the framed region of epididymis. Sense probe was used as negative control. Scale bar (left) = 500 μm, scale bar (right) = 50 μm. We monitored the Lcn6 expression during the life cycle of male mice by Northern blot and RT-PCR. It showed that Lcn6 mRNA expression started at 14 days of age, peaked at about 2-month old stage, maintained at a relatively high level in mature animals, and decreased in aged mice (Figure 2A–C). These results implied the expression of Lcn6 was developmentally regulated. Next, we investigated that whether the expression of Lcn6 was regulated by testosterone like many other epididymis-specific genes. Mice of appropriate age were sham operated, castrated 7 days or castrated but given a testosterone injection after 7 days, then the Lcn6 mRNA of epididymides was assayed by Northern blot and RT-PCR. The results showed that Lcn6 expression fell steadily after testis removal and gradually got partial recovery after testosterone supplementation, in accord with the serum testosterone level of experimental mice (Figure 2D–F). This suggests the expression of Lcn6 is partially under positive control of androgen. Figure 2. View largeDownload slide Lcn6 expression in WT mouse was developmentally and androgen regulated. Left column: (A) Northern blot and (B) RT-PCR showing mLcn6 expression in epididymis at different ages of male mice. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (C) Summary of relative mLcn6 expression (compared to loading control) during the development stages based on the Northern blot analysis. Right column: (D) Northern blot and (E) RT-PCR showing mLcn6 expression in epididymis of male mice after different days of castration and subsequent testosterone supplementation. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (F) Summary of relative mLcn6 expression (based on the Northern blot analysis) and testosterone level (compared to sham-operated control) at different days after the treatment. Figure 2. View largeDownload slide Lcn6 expression in WT mouse was developmentally and androgen regulated. Left column: (A) Northern blot and (B) RT-PCR showing mLcn6 expression in epididymis at different ages of male mice. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (C) Summary of relative mLcn6 expression (compared to loading control) during the development stages based on the Northern blot analysis. Right column: (D) Northern blot and (E) RT-PCR showing mLcn6 expression in epididymis of male mice after different days of castration and subsequent testosterone supplementation. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (F) Summary of relative mLcn6 expression (based on the Northern blot analysis) and testosterone level (compared to sham-operated control) at different days after the treatment. Establishment of conditional Lcn6 knockout mouse model To achieve inducible deletion of Lcn6, we constructed a conditional allele of the murine Lcn6 locus with loxP sites flanking exon2 (Figure 3A). The introduction of the conditional allele and removal of PGK-Neo resistance gene were confirmed by PCR using DNA from tailclippings. Lcn6 knockout mice (Lcn6Δ/Δ) were generated by TM treatment of Lcn6fl/fl; UbC-Cre/ERT2 mice at 8–12 weeks of age and were compared to TM-treated Lcn6fl/fl (control) mice. Before and after TM treatment, genotyping using DNA from tailclippings was performed to confirm the recombination events. As predicted, PCR products of the 1158-bp band representing homozygous floxed exon2 were both excised into 360-bp recombinant amplified from mice of genotype Lcn6fl/fl; UbC-Cre/ERT2, while PCR products amplified from control mice had no change under treatment (Figure 3B). The mice with complete deletion of Lcn6 genomic locus were chosen for detection of Lcn6 mRNA level. Two pairs of primers, respectively, amplifying exon2 and exon3 to exon7 were designed for qRT-PCR to evaluate the knockout efficiency. The results revealed that hardly any full-length RNA was transcribed in epididymides of Lcn6Δ/Δ mice, and the knockout efficiency was up to 90% (Figure 3C). RNA in situ hybridization analysis also confirmed the knockout of Lcn6 mRNA in epididymides of Lcn6Δ/Δ mice (Figure 3D). Therefore, the targeted disruption of Lcn6 gene was successful and the mice could be used for further examination. Figure 3. View largeDownload slide Generation of conditional Lcn6 knockout mouse model. (A) Gene targeting strategy. Mouse Lcn6 exon2 was flanked by loxP sites (red triangle), between that a Neo selection cassette was flanked by FRT sites (dark gray triangle). Black arrows indicate the primers that were used for identification of the recombination events. (B) Genotype identification of Lcn6fl/fl and Lcn6fl/fl; UbC-Cre mice before and after TM treatment. After TM, PCR fragments amplified with primer7 (P7) and primer8 (P8) were changed from 1158-bp band to 360-bp band in Lcn6fl/fl; UbC-Cre mice. (C) qRT-PCR analysis of Lcn6 mRNA level in the epididymides of Lcn6fl/fl and Lcn6Δ/Δ mice. Primers amplifying exon2 and exon3 to exon7 were used, respectively. Lcn6 expression of each mouse was first normalized to internal control (Gapdh), then relative Lcn6 mRNA level of Lcn6Δ/Δ mice was compared to that of control Lcn6fl/fl mice. Data are shown as the mean ± SEM, compared by unpaired Student t-test (two tailed). Each group consisted of 5 mice. ***P < 0.0001. (D) In situ hybridization analysis of Lcn6 mRNA level in the proximal caput of epididymides in Lcn6fl/fl and Lcn6Δ/Δ mice, respectively. Hybridization signal with the sense probe from the sample of Lcn6fl/fl mouse epididymis was used as negative control (N. C.). Scale bar = 50 μm. Figure 3. View largeDownload slide Generation of conditional Lcn6 knockout mouse model. (A) Gene targeting strategy. Mouse Lcn6 exon2 was flanked by loxP sites (red triangle), between that a Neo selection cassette was flanked by FRT sites (dark gray triangle). Black arrows indicate the primers that were used for identification of the recombination events. (B) Genotype identification of Lcn6fl/fl and Lcn6fl/fl; UbC-Cre mice before and after TM treatment. After TM, PCR fragments amplified with primer7 (P7) and primer8 (P8) were changed from 1158-bp band to 360-bp band in Lcn6fl/fl; UbC-Cre mice. (C) qRT-PCR analysis of Lcn6 mRNA level in the epididymides of Lcn6fl/fl and Lcn6Δ/Δ mice. Primers amplifying exon2 and exon3 to exon7 were used, respectively. Lcn6 expression of each mouse was first normalized to internal control (Gapdh), then relative Lcn6 mRNA level of Lcn6Δ/Δ mice was compared to that of control Lcn6fl/fl mice. Data are shown as the mean ± SEM, compared by unpaired Student t-test (two tailed). Each group consisted of 5 mice. ***P < 0.0001. (D) In situ hybridization analysis of Lcn6 mRNA level in the proximal caput of epididymides in Lcn6fl/fl and Lcn6Δ/Δ mice, respectively. Hybridization signal with the sense probe from the sample of Lcn6fl/fl mouse epididymis was used as negative control (N. C.). Scale bar = 50 μm. All the mice appeared normal and were unaffected by the introduction of loxP sites or TM treatment. When TM-treated mice were sacrificed, the weight of body, testes, and epididymides were recorded. No significant difference was found between Lcn6Δ/Δ male mice and the control Lcn6fl/fl male mice (see Supplementary Figure S3). As shown in Figure 4, histology of seminiferous tubules and epididymal structure in Lcn6Δ/Δ males did not show obvious abnormality. Tubules were filled with sperm and organization of epithelial cells in each segment of epididymides had no obvious changes. This suggests that LCN6 deficiency has no influence on the structure and organization of epididymal epithelium. Figure 4. View largeDownload slide Morphology of testis and epididymis in the Lcn6fl/fl and Lcn6Δ/Δ male mice. Images of left column showing the representative HE staining of testis and epididymis of caput, corpus, and cauda region in Lcn6fl/fl mice. Images of right column showing the representative HE staining of corresponding structure in Lcn6Δ/Δ mice. (A) and (E) Testis, (B) and (F) Caput epididymis, (C) and (G) Corpus epididymis, (D) and (H) Cauda epididymis. Regions in the dotted boxes are magnified in the bottom right corner. Scale bar (white) = 100 μm, scale bar (black) = 20 μm. Figure 4. View largeDownload slide Morphology of testis and epididymis in the Lcn6fl/fl and Lcn6Δ/Δ male mice. Images of left column showing the representative HE staining of testis and epididymis of caput, corpus, and cauda region in Lcn6fl/fl mice. Images of right column showing the representative HE staining of corresponding structure in Lcn6Δ/Δ mice. (A) and (E) Testis, (B) and (F) Caput epididymis, (C) and (G) Corpus epididymis, (D) and (H) Cauda epididymis. Regions in the dotted boxes are magnified in the bottom right corner. Scale bar (white) = 100 μm, scale bar (black) = 20 μm. Fertility test of conditional Lcn6 knockout mice Next, we performed mating test to examine natural fertility of Lcn6 null mice. During the 6-month mating, Lcn6fl/fl mating pairs gave birth to 22 liters (N = 3) while Lcn6Δ/Δ mating pairs produced 35 liters (N = 5). The number of average pups of per liter was 5.909 ± 0.4554 and 6.771 ± 0.4585, respectively. No difference was observed in the unstricted fertility test between Lcn6Δ/Δ mating pairs and Lcn6fl/fl mating pairs (Figure 5). This demonstrates that the loss of Lcn6 does not impair the fertility of male mice in vivo. Figure 5. View largeDownload slide Fertility test of Lcn6fl/fl and Lcn6Δ/Δ male mice. Numbers of pups per litter were obtained by crossing Lcn6fl/fl (N = 3) and Lcn6Δ/Δ (N = 5) male mice to normal WT female partners. Results are presented as the mean ± SEM, compared by Student t-test (two-tailed). Figure 5. View largeDownload slide Fertility test of Lcn6fl/fl and Lcn6Δ/Δ male mice. Numbers of pups per litter were obtained by crossing Lcn6fl/fl (N = 3) and Lcn6Δ/Δ (N = 5) male mice to normal WT female partners. Results are presented as the mean ± SEM, compared by Student t-test (two-tailed). Sperm functional analysis in conditional Lcn6 knockout mice Sperm in vitro fertilization ability, motility, and capacitation status Subsequently, we analyzed the cauda epididymal sperm from Lcn6Δ/Δ males in vitro. At first, sperm from Lcn6Δ/Δ males and control Lcn6fl/fl males were capacitated and subjected to in vitro fertilization. Six-hour incubation of sperm and cumulus-intact oocytes was first performed to evaluate the fertilization capability of Lcn6Δ/Δ sperm. In consequence, when adequate time was offered, Lcn6Δ/Δ sperm had the same ability as Lcn6fl/fl control sperm in fertilizing cumulus-intact eggs. We wondered whether long time compromised the dysfunction of Lcn6Δ/Δ sperm, thus 3-h incubation of sperm and cumulus-intact oocytes was implemented. However, Lcn6Δ/Δ group had the same 2-cell rate as the control group. These results demonstrate that Lcn6Δ/Δ sperm are as capable as Lcn6fl/fl sperm in fertilizing cumulus-intact eggs over a long time span (6 h) or a shorter time span (3 h) (Figure 6A). Figure 6. View largeDownload slide Unaltered sperm in vitro fertilization ability, motility, and capacitation status in Lcn6Δ/Δ mice. (A) 2-cell rates were counted 24 h after Lcn6fl/fl and Lcn6Δ/Δ cauda epididymal sperm fertilizing superovulated eggs. The rates of fertilization are shown, respectively, under incubation time of 3 h and 6 h with the eggs. All the results from independent experiments are plotted and presented as the mean ± SEM, compared by Student t-test (two-tailed). (B) Dynamic changes in motility parameters of Lcn6fl/fl and Lcn6Δ/Δ sperm during 120-min capacitation process. Each group consisted of 4 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed). VAP, average path velocity; VSL, straight-line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement. (C) Western blot analysis of protein tyrosine phosphorylation in Lcn6fl/fl and Lcn6Δ/Δ sperm during capacitation in the indicated time. The expression of α-tubulin was used as loading control. Figure 6. View largeDownload slide Unaltered sperm in vitro fertilization ability, motility, and capacitation status in Lcn6Δ/Δ mice. (A) 2-cell rates were counted 24 h after Lcn6fl/fl and Lcn6Δ/Δ cauda epididymal sperm fertilizing superovulated eggs. The rates of fertilization are shown, respectively, under incubation time of 3 h and 6 h with the eggs. All the results from independent experiments are plotted and presented as the mean ± SEM, compared by Student t-test (two-tailed). (B) Dynamic changes in motility parameters of Lcn6fl/fl and Lcn6Δ/Δ sperm during 120-min capacitation process. Each group consisted of 4 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed). VAP, average path velocity; VSL, straight-line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement. (C) Western blot analysis of protein tyrosine phosphorylation in Lcn6fl/fl and Lcn6Δ/Δ sperm during capacitation in the indicated time. The expression of α-tubulin was used as loading control. It is well known that ejaculated sperm have to swim through the female reproductive tract and capacitate there before fertilizing eggs in vivo. Thus, sperm motility and capacitation status are important parameters for fertilization potential. We assessed the motility of cauda epididymal sperm of Lcn6Δ/Δ and control mice by a computer-assisted sperm analysis system. During the whole 120-min process of capacitation in BWW medium, the total motility and progressive motility were still indistinguishable between control and Lcn6 null mouse sperm. Other motility parameters including average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) were slightly elevated in Lcn6Δ/Δ sperm through the capacitation process, though without significant statistical significance (Figure 6B). A distinguishing feature associated with sperm capacitation is the increase in protein tyrosine phosphorylation. Thus tyrosine phosphorylation of sperm proteins was monitored by Western blot during the capacitation process. We found Lcn6Δ/Δ sperm had the normal protein tyrosine phosphorylation showing gradual increase over time as the control sperm did (Figure 6C). Sperm acrosome reaction AR is important for sperm to fertilize the egg. Therefore, the ability of cauda epididymal sperm from Lcn6Δ/Δ males to undergo AR was assessed. When non-capacitated or capacitated for 1 h, Lcn6Δ/Δ sperm showed an enhanced spontaneous AR frequencies in comparison to control (P < 0.01) (Figure 7A; see representative pictures in Supplementary Figure S4). It suggests the deficiency of LCN6 protein leads to disordered spontaneous AR of sperm. To further investigate the capability of Lcn6Δ/Δ sperm in induced AR, capacitated sperm from the Lcn6Δ/Δ and control mice were, respectively, treated with Ca2+ ionophore A23187 and subjected to the AR frequency assessment. Nevertheless, no statistical difference was found between the Lcn6Δ/Δ sperm and the control (Figure 7A; see representative pictures in Supplementary Figure S4). It indicates Lcn6Δ/Δ sperm show the same response to A23187 as the control do. Figure 7. View largeDownload slide Impaired sperm spontaneous AR and intracellular calcium level in Lcn6Δ/Δ mice. (A) Comparison of sperm spontaneous and Ca2+ ionophore A23187 induced AR frequency between Lcn6fl/fl and Lcn6Δ/Δ male mice. Sperm spontaneous AR frequencies are, respectively, shown in the capacitation status (1 h) or not (0 h). Induced AR frequencies are obtained from sperm capacitated for 1 h. Each group consisted of at least 3 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed), **P < 0.01. (B) Comparison of intracellular calcium level of Lcn6fl/fl and Lcn6Δ/Δ sperm without capacitation (left panel) or under 1 h capacitation (right panel) in the indicated medium. BWW-Ca, BWW buffer without calcium. Each group consisted of 4 mice, data are shown as the mean ± SEM, compared by Student t-test (two-tailed). **P < 0.01. Figure 7. View largeDownload slide Impaired sperm spontaneous AR and intracellular calcium level in Lcn6Δ/Δ mice. (A) Comparison of sperm spontaneous and Ca2+ ionophore A23187 induced AR frequency between Lcn6fl/fl and Lcn6Δ/Δ male mice. Sperm spontaneous AR frequencies are, respectively, shown in the capacitation status (1 h) or not (0 h). Induced AR frequencies are obtained from sperm capacitated for 1 h. Each group consisted of at least 3 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed), **P < 0.01. (B) Comparison of intracellular calcium level of Lcn6fl/fl and Lcn6Δ/Δ sperm without capacitation (left panel) or under 1 h capacitation (right panel) in the indicated medium. BWW-Ca, BWW buffer without calcium. Each group consisted of 4 mice, data are shown as the mean ± SEM, compared by Student t-test (two-tailed). **P < 0.01. Sperm AR is under precise control of numerous factors, of which calcium is the most important. Thus, we measured the intracellular calcium level of Lcn6 null sperm by using calcium indicator Fura2-AM. The intracellular calcium level in complete BWW medium was first analyzed. As shown in Figure 7B, whether sperm were capacitated for 1 h or not, Lcn6Δ/Δ sperm exhibited elevated intracellular calcium levels compared to control (P < 0.01). Then we wanted to know whether the increased calcium level of the Lcn6Δ/Δ sperm could be influenced by the extracellular calcium. Sperm from the Lcn6Δ/Δ and control mice were released into BWW medium without calcium, and then subjected to analysis of intracellular calcium level. Surprisingly, no difference can be detected when calcium was depleted from the external medium (Figure 7B). All these results suggest that LCN6 deficiency leads to impaired intracellular calcium balance of sperm. Discussion Previous studies from us and other groups have shown the involvement of lipocalins in sperm function in vitro [13–15,17]. However, these reports fail to elucidate their physiological roles in vivo. Our present study for the first time investigates and identifies the in vivo function of LCN6, an epididymis-specific lipocalin protein. It might be helpful to shed light on the physiological roles of epididymal lipocalins. As mentioned before, we suppose the generation of the animal model specifically deleting Lcn6 gene in adulthood might avoid possible complementary mechanism of molecular function during epididymal development. This advantage is supported by the unaltered expression of other lipocalins in epididymides of Lcn6 null mice (see Supplementary Figure S5). Nevertheless, the disadvantage of failing to reveal importance of individual gene when cluster of genes have some redundancy in their function is the same as that of other conventional knockout strategies. We speculate that the unchanged fertilization capacity of conditional Lcn6 knockout mice after Lcn6 deficiency (Figure 5) could be attributed to that. The epididymis-specific lipocalins are so important for male fertility, therefore functional substitution might arise during fertilization. This can somewhat be suggested by the existence of the epididymis-specific lipocalin cluster and their similar features of dependence on testicular factors and androgen [10,12,16,37]. From this point of view, it might be of great value to delete this cluster of lipocalins in the future to investigate their specific function in epididymis. However, we cannot exclude the possibility that mouse LCN6 might function in the development of epididymis since we only investigate the influence of LCN6 deficiency on adult mice. Our future work will focus on that. Though the unaltered male fertility of adult Lcn6 null mice, it is deserved to be mentioned that this mouse model is a meaningful attempt to targeted disruption of epididymis-specific gene in time-space manner. Several Cre recombinase transgenic mice that express the Cre recombinase under the control of epididymis-specific genes (e.g. Crisp4 [29], Defb41 [28], Lcn5 [38], and Rnase10 [39]) had been constructed to accomplish targeted elimination of genes. The benefits of these Cre mouse lines are obvious, so are the drawbacks. In these models, gene inactivation recombination event only occurs in particular regions at specialized time, of which most is before puberty. For example, the Cre recombinase expression of Crisp4-Cre, Defb41-Cre, and Rnase10-Cre mice started at Postnatal Day 20, and of the Lcn5-Cre mouse, it initiated at Day 30. Cre activity in all of these mouse lines is confined to the caput epididymis. Nevertheless, sperm maturation happens after sexual maturity and many genes specifically expressed in the corpus and cauda epididymis are actively involved in the maturation process. Therefore, conventional knockout strategies in epididymal research using existing Cre mouse lines are unlikely to meet the demand. In the present study, we accomplished a nearly complete deletion of Lcn6 in adult epididymis by using an extensively expressed and TM induced Cre line. And we can even delete Lcn6 gene in childhood of the mice if necessary. Furthermore, it is the first case that a TM induced knockout mouse model has been established in the epididymal research. During this process, we have not found any visible side effects on male fertility under this dosage of TM. But we are honest to say that TM is a kind of anti-estrogen drug and other researchers on male fertility should take this into consideration and observe its effects seriously when using it. We characterized two transcript variants of mouse Lcn6 that were specifically detected in the epididymis among all the tested mouse tissues. However, a sole transcript was found in the epididymis of human and rat [40]. Based on that, we wondered whether each of the mouse Lcn6 transcripts had its own function. Thus, we made a great effort on the production of anti-mouse LCN6α and LCN6β antibody. Unfortunately, all the antibodies, whether commercial or produced by ourself, failed to detect the in vivo LCN6 protein. Therefore, the respective function of mouse LCN6 isoforms is not able to be exposed. When orchidectomy and testosterone injections were performed in WT epididymis, the changes in the mRNA level of Lcn6 suggest its regulation by androgen. But its expression not getting complete recovery after testosterone replacement (Figure 2) indicates that other factors in testicular fluid are involved. It has long been proved that testosterone levels restoration could reverse changes in the caput, corpus, and cauda epididymis after orchidectomy, but not in the initial segment [41]. This is in accordance with the fact that Lcn6 mRNA exists mostly in the initial segment and some in the other parts of caput epididymis (Figure 1). We speculate that LCN6 protein is regulated by testicular factors as well as androgen and it possesses important functions in the epididymis. When both transcripts are null in Lcn6 knockout mice, no obvious change could be observed in the morphology of epididymides (Figure 4), while cauda epididymal sperm from the Lcn6 knockout mice indeed have a premature AR (Figure 7). This demonstrates the function of LCN6 on regulation of sperm AR. Furthermore, our in vitro experimental results show that only mLCN6α is inclined to be secreted into extracellular environment when 3× flag-tagged mLCN6α and mLCN6β protein were over-expressed in HEK293T cell, respectively or simultaneously. The flag fusion mLCN6α protein even can be incorporated onto/into Lcn6 deficient sperm head, exactly between the equatorial segment and the post-acrosomal region (see Supplementary Figure S6). These indicate that LCN6 protein, especially LCN6α, might help to prevent the happening of premature AR. To fuse with oocytes, mammalian sperm must pass through the cumulus cell layer and the zona pellucida (ZP) of eggs. At this point, the AR is initiated [42–44]. The acrosomal reaction normally takes place in the present of ovum. But a certain percentage of sperm undergo spontaneous AR without the presence of oocytes [45]. This fundamental maturational process is regulated by numerous signaling cascades, and calcium plays a dynamic role in this process [46,47]. It is known that a high and sustained calcium influx is necessary for the AR to occur and Ca2+ ionophore that elevates intracellular calcium level can increase the AR frequency [48–50]. In fact, the ability to undergo Ca2+ ionophore A23187 induced AR could be used as a clinical marker to evaluate the fertilization potential of sperm [51–53]. In Lcn6 deficient sperm, spontaneous AR frequency is higher than control sperm whether capacitated or not, but Ca2+ ionophore A23187 induced AR frequency has no significant change (Figure 7). These evidences imply that LCN6 deficiency leads to impaired sperm maturation in AR to a certain extent, without harm to the fertilization potential of the sperm. However, the elevation of spontaneous AR frequency is in accompany with the increase of intracellular calcium level in Lcn6 knockout sperm, and that rise of intracellular calcium level depends on the calcium in the medium (Figure 7). This is consistent with the fact that spontaneous AR is strictly dependent on the presence of extracellular calcium [54]. These evidences indicate that LCN6 deficiency leads to the rise of calcium influx in the sperm and possibly LCN6 protein is involved in the regulation of calcium influx. Cauda epididymal sperm from the Lcn6 null mouse still gain the ability to fertilize the egg in vitro (Figure 6). Though this is in conformity with their phenotype of nonimpaired Ca2+ ionophore A23187 induced AR frequency, it is generally considered that sperm in the early stages of AR may bind to and go on to penetrate the ZP, but those that undergo AR prematurely will be severely compromised in their ability to fertilize [55]. As a matter of fact, Jin et al. investigated the mouse sperm AR status before and during the fertilizing process, it turned out that most fertilizing sperm underwent the AR before reaching the ZP of cumulus-enclosed oocytes [56]. Inoue et al. even demonstrated the possibility of that acrosome-reacted mouse sperm recovered from the perivitelline space can fertilize other eggs [57]. Based on these evidence, we consider our investigation on Lcn6 deficient sperm provides further evidence for the theory of that premature AR in a certain range is nonlethal to the in vitro fertilization potential of sperm. When it comes to enhanced spontaneous AR’s impact on in vivo fertilization, it seems less optimistic. Considerable references show that ejaculated sperm have a basic low level of spontaneous AR rate until they get close enough to the ovulated eggs [58–60]. That means an impaired fecundity should be observed in Lcn6 null mice when their sperm exhibit premature AR. But in fact, it is not the case. In addition to the above mentioned possible functional redundancy of lipocalins, we speculate this phenomenon could be interpreted from the other perspective. Functional substitution is not always limited to the same gene family although in most case it is [61,62]. In fact, CD46 deficient mice exhibited similar phenotype as the Lcn6 null mice, which had an accelerated spontaneous AR of sperm and increased male fertility [63]. That indicates the significance of sperm-oocyte interaction and existence of cumulatively functioning genes in the course. So there is still a possibility that non-lipocalins might substitute for the lost of LCN6 function, which could give rise to the unaltered fecundity of Lcn6 null mice. In summary, we conclude that mouse Lcn6 is not required for male fecundity, but has an irreplaceable role in sperm maturation. Supplementary data Supplementary data are available at BIOLRE online. Supplemental information contains Supplemental Experimental Procedures, six figures, and one table that are available online. Supplementary Figure S1. The location of Lcn6 gene on mouse chromosome, with the arrangement of other lipocalin genes in the gene cluster. Supplementary Figure S2. The cDNA and amino acid sequence of two isoforms of mLcn6. Supplementary Figure S3. (A) Photography of testes and epididymides of Lcn6fl/fl and Lcn6Δ/Δ male mice. (B) Comparison of the ratio of testis/body weight and epididymis/body weight between Lcn6fl/fl and Lcn6Δ/Δ male mice. Supplementary Figure S4. Representative pictures in assessment of spontaneous AR frequency and Ca2+ ionophore A23187 induced AR frequency in Lcn6 null mice and control Lcn6fl/fl mice. Supplementary Figure S5. qRT-PCR analysis of mRNA level of other lipocalin genes in Lcn6 null mice and control Lcn6fl/fl mice. Supplementary Figure S6. Expression of 3× flag-tagged mLCN6α protein and its incorporation onto/into Lcn6 deficient sperm. Supplementary Table S1. Primers that were used in the manuscript. Author Contributions: Jia Shen prepared Figures 1 and 2 and Qianqian Yin prepared Figures 3–7. Xiaofeng Wang constructed the animal model. 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Impaired sperm maturation in conditional Lcn6 knockout mice

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Abstract

Abstract Human LCN6, a lipocalin protein, exhibits predominant expression in epididymis and location on the sperm surface. However, the biological function of LCN6 in vivo remains unknown. Herein, we found that unlike human LCN6, mouse Lcn6 gene encoded two transcript variants that were both upregulated by androgen. Subsequently, we generated a conditional knockout mouse model to disrupt Lcn6 in the adult and investigate its function. In this model, spermatogenesis was normal and Lcn6 deficiency did not affect the natural birth rate of male mice or in vitro fertilization ability of their cauda epididymal sperm. Nevertheless, sperm from the cauda epididymis of the Lcn6 null mice underwent a sustained increase of acrosome reaction frequency whether capacitated or not (P < 0.01). Consistent with premature acrosome reaction, sperm from knockout mice had significantly increased intracellular calcium content when extracellular calcium was supplied (P < 0.01). These results demonstrate an important function of LCN6 in preventing calcium overload and premature acrosome reaction of sperm and suggest a potential risk factor of LCN6 deficiency for sperm maturation. Introduction Sperm accomplishing the developmental stage in the testis are immotile and immature. They gain progressive motility and undergo maturation during their transit through epididymis. This maturation process is thought to be completed in the interaction between sperm and proteins synthesized and secreted by the epididymal epithelium [1–3]. The mammalian epididymis is divided into three regions: caput, corpus, and cauda. In fact, the most proximal caput region of rodents’ epididymides exhibits a feature distinct from the other caput region, thus, this special part is named the initial segment. Each segment of epididymis has its unique gene expression patterns, leading to that diverse secretory proteins function in sperm maturation sequentially [4–6]. But the specific roles of these epididymal proteins, especially that exclusively or predominantly expressed in epididymis, are largely unknown. Lipocalin family is an ancient superfamily of extracellular proteins that function in a broad range of systems including taste and odor chemoreception and transport, coloration, immune modulation, prostaglandin D synthesis, and metabolism [7–12]. Its involvement in sperm maturation was highlighted by several reports. LCN2, also known as 24p3, was reported to act as a suppressor of acrosome reaction (AR) [13], and to enhance sperm motility through elevation of intracellular pH and increase of intracellular cyclic adenosine monophosphate accumulation [14]. LCN5, the epididymal retinoic acid binding protein, was indicated to function in the maintenance of epithelium of cauda epididymis [15]. However, the understanding about lipocalins on male fertility is far from complete. In mouse and human, there is a highly conserved lipocalin gene cluster with similar gene number, order, and orientation. Within the cluster, Lcn5, Lcn8, Lcn9, Lcn10, Lcn12, and Lcn13 were first cloned and proved specifically expressed in the mouse epididymis [16]. Previously, we cloned a novel lipocalin gene named Lcn6 that is adjacent to Lcn5 and Lcn8 (see Supplementary Figure S1). The encoded protein in human was predominantly located in epididymis and associated with the sperm head and neck [17], suggesting the importance of LCN6 in male fertility. Conventional knockout mouse models have been widely used in the functional research for epididymal proteins. For example, cSrc, transaldolase, and c-ros receptor tyrosine kinase were demonstrated their importance in epididymal development and sperm maturation [18–20]. But it is noteworthy that a number of mice with deletion of epididymis-specific genes generated by conventional knockout approaches do not show fertility failure phenotype [21–23]. This phenomenon can be attributed to some developmental compensatory mechanism that is common in other organs and species and thought to be created during the evolution [24–26]. In addition, a recent work describing that homozygous deletion of a cluster of nine β-defensin genes in the mouse epididymis results in male sterility sincerely supports the existence of compensation [27]. For this reason, establishment of conditional gene null mouse models to elucidate the epididymal function is required. Nevertheless, there are scarce research papers using conditional knockout mouse model to research on epididymis [28,29], and induced gene disruption in adult epididymis has not been reported. Here, we established a knockout mouse model in which Lcn6 deletion was achieved in the adult mice by tamoxifen (TM) administration to investigate the in vivo function of LCN6 on male fertility. Materials and methods Animals, tissue preparation All the experimental procedures were carried out according to the protocol with the approval of the Institutional Animal Care and Use Committee (approved number: SIBCB-S281-1510-2-041). Mice of the wild-type (WT) strain C57BL/6 were supplied by the Animal Center of the Chinese Academy of Sciences (Shanghai, China). Prior to dissection, animals were euthanized with CO2 inhalation. Tissues for mRNA analysis were excised and frozen immediately in liquid nitrogen, tissues for in situ hybridization and Hematoxylin and Eosin (HE) staining were fixed in 4% paraformaldehyde (PFA) or Bouin fluid for further process. Castration and androgen replacement 8-week-old WT male mice were castrated bilaterally under sodium pentobarbital anesthesia. Animals were divided into 9 groups (3–6 mice per group), and killed on Day 0, 1, 3, 5, 7 after castration as well as 1, 3, 5, 7 days after the initial testosterone propionate injection. Androgen supplementation began on the seventh day after castration at the dose of 4 mg/kg body weight. Changes in the serum testosterone levels of each group were confirmed by radioimmunoassay in Shanghai Zhongshan Hospital. Total RNA isolation and Northern blot analysis Total RNA was extracted from tissue homogenates with TRIzol reagents (Invitrogen, USA) according to the manufacturer's instructions. Northern blot analysis was performed as described previously [30]. Briefly, 20 μg of total RNA from each sample was subjected to 1.2% (w/v) agarose-formaldehyde gel electrophoresis, blotted onto nylon membranes by capillary transfer, and hybridized with a probe that was a 32P-labeled cDNA fragment of mouse Lcn6. The hybridization signal from 18S rRNA was used as loading control. Autoradiographs with pronounced differences in expression were analyzed by densitometry. The probes are amplified from epididymal cDNA using the primers listed in Supplementary Table S1. Reverse transcription polymerase chain reaction (RT-PCR) and Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) The first strand of cDNA was synthesized with a reverse transcription (RT) kit (Takara, FSQ-301) from mRNA extract. Then the products were diluted 25-fold and subjected to PCR using reagents of 2× Taq PCR Master Mix from Takara (Japan). qRT-PCR was performed with 2× SYBR Green qPCR Master Mix from Bimake (USA) on an ABI QuantStudio 6 Flex Real-Time PCR Detection System (Applied Biosystems, USA). All the primers are listed in Supplementary Table S1. In situ hybridization In situ hybridization of Lcn6 mRNA in WT epididymis was performed following the protocol previously described [31] with a few modifications. Firstly, a fragment of corresponding Lcn6α cDNA was correctly cloned into the pGEMT-Easy Vector (Promega, A1360). Secondly, the digoxigenin-labeled probe RNA was synthesized through in vitro transcription with SP6 and T7 RNA polymerase using DIG RNA labeling Kit (Roche, 1175025). Thirdly, after incubation of epididymal sections and digoxigenin-labeled probe, alkaline phosphatase coupled with anti-digoxigenin antibody (Roche, 11093274910) was used and the in situ signal was developed with NBT/BCIP system (Roche, 11681451001). The sense probe was used as negative control. Primers that were used to amplify Lcn6α are listed in Supplementary Table S1. Generation of conditional Lcn6 knockout mice The 4.4 kb upstream and 3.5 kb downstream homologous arms flanking exon 2 were amplified by PCR from 129/Sv mouse genomic DNA. These fragments were cloned into the vector that had a backbone for the targeting replacement. We used PBR322 with the neomycin resistance gene (Neo) flanked by flippase recognition target (FRT) sites and loxP sites and unique restriction sites to introduce the targeted exon and the homologous arms. The final vector was electroporated into embryonic stem cells, and correctly targeted clones were screened by PCR for homologous recombination. Then chimeric mice with the genotype Lcn6fl/+; neo/+ were generated and crossed to a Flp line to obtain Neo resistance gene deletion mice carrying the Lcn6 conditional alleles (Lcn6fl/+). Subsequently, Lcn6fl/+ mice were crossed to mice expressing a TM-inducible Cre-recombinase, which is under control of the Ubiquitin C promoter, referred to as UbC-Cre/ERT2. The offsprings with the genotype Lcn6fl/+; UbC-Cre/ERT2 were backcrossed to Lcn6fl/+ to derive Lcn6fl/fl; UbC-Cre/ERT2 mice and Lcn6fl/fl mice. The Lcn6fl/fl; UbC-Cre/ERT2 offsprings obtained from the parental Lcn6fl/fl; UbC-Cre/ERT2 male mice by Lcn6fl/fl cross were for further experiments, while male Lcn6fl/fl littermates were served as controls. Cre-ERT2 recombinase was activated in adult mice at 8–12 week of age by administering TM dissolved in Corn Oil (Sigma). Mice were injected i.p. every other day for 5 days at a concentration of 2 mg/mouse (∼77 mg/kg). The day of the first injection was marked as day 0, two weeks after TM administration, TM-treated males were, respectively, caged with WT female mice at the ratio of 1:2 for two rounds (one week for a round) to evacuate sperm that already existed. Then further analyses were applied. The primers used for genotyping are listed in Supplementary Table S1. Hematoxylin and Eosin staining Fixed testes and epididymides were dehydrated using graded ethanol (0, 30%, 50%, 75%, 85%, 95%, and 100%), vitrified by dimethylbenzene and embedded in the paraffin. Then the paraffin embedding epididymides were cut into sections with thickness of 5 μM. Finally, HE staining was performed by standard procedures. Fertility assay Eligible TM-treated males were mated with 6-week-old females at the ratio of 1:2 for 6 months. During this period, the size and sex ratio of every litter were recorded. Assessment of cauda epididymal sperm motility and tyrosine phosphorylation All the procedures were performed as reported previously [32] with minor modifications. In general, the cauda epididymal sperm were released into “Biggers, Whitten, and Whittingham” (BWW) medium [33] at an appropriate concentration of 5–10×106 cells/ml. This time-point was defined as the beginning of capacitation (0 min), then the sperm were capacitated for various time periods. During the whole capacitation process (120 min), the sperm motility was assessed using a computer-assisted semen analysis machine, the sperm extracts were collected and subjected to Western blot for tyrosine phosphorylation analysis. Western blot analysis Proteins were extracted from sperm by directly mixing with 1× laemmli loading buffer (Sigma) and boiled for 10 min at 100°C. Then they were resolved by SDS/polyacrylamide gel electrophoresis using a 4% stacking gel and a 12% separating gel and transferred to a PVDF membrane (Amersham/GE). After blocking overnight, the membranes were incubated with the primary antibodies and secondary antibodies listed below to detect the protein expression. The following antibodies were used: anti-phosphotyrosine monoclonal antibody, clone 4G10 (Merck/millipore, 1:10 000), anti-α-tubulin monoclonal antibody (Sigma, 1:20 000), Goat anti-mouse IgG, H&L Chain Specific Peroxidase Conjugate (Merck/millipore, 1:10 000). In vitro fertilization In vitro fertilization was performed as described elsewhere [34] with some modifications. Briefly, 3-week-old WT female mice were superovulated and cumulus-intact oocytes were collected. Then oocytes were pooled and divided into several groups. The sperm from TM-treated males were added to the fertilization droplet containing the eggs. After 3 h or 6 h incubation, the eggs were washed to remove unbound sperm and transferred to new fertilization droplets. Twenty-four hours later, fertilization rates were evaluated by recording the number of two-cell embryo. Evaluation of sperm acrosome reaction frequency The sperm AR frequency was assessed as described elsewhere [35]. In Brief, released sperm were capacitated for the indicated time, centrifuged and washed with PBS, then spreaded onto slides, airdried and fixed with 4% PFA. Then 3 μM final concentration of fluorescein isothiocyanate conjugated lectin from Arachis hypogaea (peanut) (Sigma) was used to stain the acrosome of spermatozoa, and DNA was counterstained with 4΄,6-diamidino-2-phenylindole (Sigma). To assess the Ca2+ ionophore A23187 induced AR frequency, released sperm were capacitated for 1 h and treated with 10 μM A23187 (Sigma) for 15 min, then the above procedures were performed. Measurement of [Ca2+]i concentration The measurement of sperm intracellular calcium level was carried out as described elsewhere [36]. Spermatozoa were allowed to disperse into the BWW medium without Ca2+ or complete BWW as indicated and loaded with the acetoxy-methyl ester of fura-2 (Fura-2/AM; Sigma; 3 μM) according to the manufacturer's protocol. The dynamic range for Ca2+-dependent fluorescence signals was obtained on a BioTek Synergy NEO multifunctional microplate detector by using excitation at 340 nm and 380 nm and ratioing the fluorescence in tensities detected at ∼510 nm. The [Ca2+] i was calculated using the equation $$\,\mathop {[ {{\rm{C}}{{\rm{a}}^{{\rm{2 + }}}}} ]}\nolimits_i = {K_d}{{( {F - {F_{\min }}} )} / {( {{F_{\max }} - F} )}}$$, where Kd = 224 nM, $$F = {{{F_{340}}} / {{F_{380}}}}$$. Fmax and Fmin were recorded at the end of the incubation period after supplementation of calcium when extracellular calcium was depleted. Fmax was determined after the addition of 20 mM digitonin, and Fmin was determined after addition of 10 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N΄,N΄-tetraacetic acid, of which the pH was adjusted to 8.4 with Tris base. Each sample was measured in triplicate. Images acquisition and processing All the slides were observed and photographed under an Olympus BX51 fluorescent microscope equipped with a DP70 camera. Images to evaluate the AR frequency were processed by Image-Pro Plus (version 6.0) and then determined by manual count. At least 200 sperm were scored in each sample. Statistical analysis All of the Western blot and Northern blot images and RT-PCR results are representative of at least three independent experiments. The qRT-PCR was performed in triplicate. Data were analyzed by Prism software (version 6.01) and P < 0.05 was defined as statistically significant. Results Epididymis-specific and androgen-regulated expression of the mouse Lcn6 The mouse ortholog of human LCN6 was obtained by homology search of the mouse expressed sequence tag database at http://www.ncbi.nlm.nih.gov/BLAST based on human and rat Lcn6 cDNA sequence. The full-length cDNA of mouse Lcn6 was amplified by RT-PCR and sequenced. Unlike human LCN6, mouse Lcn6 gene encodes two transcripts named after mLcn6α and mLcn6β, respectively (Figure 1A). Both transcripts have 7 exons and 6 introns characteristic of lipocalin gene family, and they share the exact same sequence ahead of exon6. But due to alternative splicing, variant mLcn6β skips the stop codon that mLcn6α has and encodes a longer isoform than mLcn6α (Figure 1A, top panel; see Supplementary Figure S2). As predicted with SignalP 4.1 server at http://www.cbs.dtu.dk/services/SignalP, the N-terminal 21 amino acid of Lcn6 encoded proteins probably formed a signal peptide (Figure 1A, bottom panel; see Supplementary Figure S2). In the male mice, both transcripts were exclusively detected in the epididymis rather than other reproductive organs such as testis, seminal vesicle, prostate and vas deferens and other male organs (Figure 1B, left panel). While in the female, neither of them was detected in any tested tissues (Figure 1C). A further examination by Northern blot hybridization showed the specific expression of Lcn6 in the caput but not in the other portions of epididymis (Figure 1B, right panel). Moreover, using RNA in situ hybridization we confirmed the localization of Lcn6 transcripts was mainly in the proximal caput epididymis (Figure 1D). Figure 1. View largeDownload slide Schematic diagram of mLcn6 transcript variants and expression pattern of mLcn6 in WT mouse. (A) Structure of pre-mRNAs (top panel) and encoded proteins (bottom panel). Top panel: box, exon; line, intron; number indicates the length of exon or intron. Bottom panel: box, amino acid sequence; number indicates where it starts and ends. (B) Left panel: Northern blot analysis of the mLcn6 expression in 16 tissues of male mice and rat epididymis. Right panel: Northern blot analysis of mLcn6 expression in precise segments of mouse epididymis. Template from testis was used as negative control. 18S rRNA was used as loading control. (C) RT-PCR analysis of mLcn6 expression in 9 tissues from female mice. Template from mouse epididymis and template without cDNA were used as positive control and negative control (N. C.), respectively. Gapdh mRNA was used as loading control. (D) In situ hybridization analysis of Lcn6 mRNA localization (blue) in the caput epididymis. Left panel shows the full picture, right panel shows the magnified picture of the framed region of epididymis. Sense probe was used as negative control. Scale bar (left) = 500 μm, scale bar (right) = 50 μm. Figure 1. View largeDownload slide Schematic diagram of mLcn6 transcript variants and expression pattern of mLcn6 in WT mouse. (A) Structure of pre-mRNAs (top panel) and encoded proteins (bottom panel). Top panel: box, exon; line, intron; number indicates the length of exon or intron. Bottom panel: box, amino acid sequence; number indicates where it starts and ends. (B) Left panel: Northern blot analysis of the mLcn6 expression in 16 tissues of male mice and rat epididymis. Right panel: Northern blot analysis of mLcn6 expression in precise segments of mouse epididymis. Template from testis was used as negative control. 18S rRNA was used as loading control. (C) RT-PCR analysis of mLcn6 expression in 9 tissues from female mice. Template from mouse epididymis and template without cDNA were used as positive control and negative control (N. C.), respectively. Gapdh mRNA was used as loading control. (D) In situ hybridization analysis of Lcn6 mRNA localization (blue) in the caput epididymis. Left panel shows the full picture, right panel shows the magnified picture of the framed region of epididymis. Sense probe was used as negative control. Scale bar (left) = 500 μm, scale bar (right) = 50 μm. We monitored the Lcn6 expression during the life cycle of male mice by Northern blot and RT-PCR. It showed that Lcn6 mRNA expression started at 14 days of age, peaked at about 2-month old stage, maintained at a relatively high level in mature animals, and decreased in aged mice (Figure 2A–C). These results implied the expression of Lcn6 was developmentally regulated. Next, we investigated that whether the expression of Lcn6 was regulated by testosterone like many other epididymis-specific genes. Mice of appropriate age were sham operated, castrated 7 days or castrated but given a testosterone injection after 7 days, then the Lcn6 mRNA of epididymides was assayed by Northern blot and RT-PCR. The results showed that Lcn6 expression fell steadily after testis removal and gradually got partial recovery after testosterone supplementation, in accord with the serum testosterone level of experimental mice (Figure 2D–F). This suggests the expression of Lcn6 is partially under positive control of androgen. Figure 2. View largeDownload slide Lcn6 expression in WT mouse was developmentally and androgen regulated. Left column: (A) Northern blot and (B) RT-PCR showing mLcn6 expression in epididymis at different ages of male mice. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (C) Summary of relative mLcn6 expression (compared to loading control) during the development stages based on the Northern blot analysis. Right column: (D) Northern blot and (E) RT-PCR showing mLcn6 expression in epididymis of male mice after different days of castration and subsequent testosterone supplementation. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (F) Summary of relative mLcn6 expression (based on the Northern blot analysis) and testosterone level (compared to sham-operated control) at different days after the treatment. Figure 2. View largeDownload slide Lcn6 expression in WT mouse was developmentally and androgen regulated. Left column: (A) Northern blot and (B) RT-PCR showing mLcn6 expression in epididymis at different ages of male mice. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (C) Summary of relative mLcn6 expression (compared to loading control) during the development stages based on the Northern blot analysis. Right column: (D) Northern blot and (E) RT-PCR showing mLcn6 expression in epididymis of male mice after different days of castration and subsequent testosterone supplementation. 18S rRNA and Gapdh mRNA were used as loading control, respectively. (F) Summary of relative mLcn6 expression (based on the Northern blot analysis) and testosterone level (compared to sham-operated control) at different days after the treatment. Establishment of conditional Lcn6 knockout mouse model To achieve inducible deletion of Lcn6, we constructed a conditional allele of the murine Lcn6 locus with loxP sites flanking exon2 (Figure 3A). The introduction of the conditional allele and removal of PGK-Neo resistance gene were confirmed by PCR using DNA from tailclippings. Lcn6 knockout mice (Lcn6Δ/Δ) were generated by TM treatment of Lcn6fl/fl; UbC-Cre/ERT2 mice at 8–12 weeks of age and were compared to TM-treated Lcn6fl/fl (control) mice. Before and after TM treatment, genotyping using DNA from tailclippings was performed to confirm the recombination events. As predicted, PCR products of the 1158-bp band representing homozygous floxed exon2 were both excised into 360-bp recombinant amplified from mice of genotype Lcn6fl/fl; UbC-Cre/ERT2, while PCR products amplified from control mice had no change under treatment (Figure 3B). The mice with complete deletion of Lcn6 genomic locus were chosen for detection of Lcn6 mRNA level. Two pairs of primers, respectively, amplifying exon2 and exon3 to exon7 were designed for qRT-PCR to evaluate the knockout efficiency. The results revealed that hardly any full-length RNA was transcribed in epididymides of Lcn6Δ/Δ mice, and the knockout efficiency was up to 90% (Figure 3C). RNA in situ hybridization analysis also confirmed the knockout of Lcn6 mRNA in epididymides of Lcn6Δ/Δ mice (Figure 3D). Therefore, the targeted disruption of Lcn6 gene was successful and the mice could be used for further examination. Figure 3. View largeDownload slide Generation of conditional Lcn6 knockout mouse model. (A) Gene targeting strategy. Mouse Lcn6 exon2 was flanked by loxP sites (red triangle), between that a Neo selection cassette was flanked by FRT sites (dark gray triangle). Black arrows indicate the primers that were used for identification of the recombination events. (B) Genotype identification of Lcn6fl/fl and Lcn6fl/fl; UbC-Cre mice before and after TM treatment. After TM, PCR fragments amplified with primer7 (P7) and primer8 (P8) were changed from 1158-bp band to 360-bp band in Lcn6fl/fl; UbC-Cre mice. (C) qRT-PCR analysis of Lcn6 mRNA level in the epididymides of Lcn6fl/fl and Lcn6Δ/Δ mice. Primers amplifying exon2 and exon3 to exon7 were used, respectively. Lcn6 expression of each mouse was first normalized to internal control (Gapdh), then relative Lcn6 mRNA level of Lcn6Δ/Δ mice was compared to that of control Lcn6fl/fl mice. Data are shown as the mean ± SEM, compared by unpaired Student t-test (two tailed). Each group consisted of 5 mice. ***P < 0.0001. (D) In situ hybridization analysis of Lcn6 mRNA level in the proximal caput of epididymides in Lcn6fl/fl and Lcn6Δ/Δ mice, respectively. Hybridization signal with the sense probe from the sample of Lcn6fl/fl mouse epididymis was used as negative control (N. C.). Scale bar = 50 μm. Figure 3. View largeDownload slide Generation of conditional Lcn6 knockout mouse model. (A) Gene targeting strategy. Mouse Lcn6 exon2 was flanked by loxP sites (red triangle), between that a Neo selection cassette was flanked by FRT sites (dark gray triangle). Black arrows indicate the primers that were used for identification of the recombination events. (B) Genotype identification of Lcn6fl/fl and Lcn6fl/fl; UbC-Cre mice before and after TM treatment. After TM, PCR fragments amplified with primer7 (P7) and primer8 (P8) were changed from 1158-bp band to 360-bp band in Lcn6fl/fl; UbC-Cre mice. (C) qRT-PCR analysis of Lcn6 mRNA level in the epididymides of Lcn6fl/fl and Lcn6Δ/Δ mice. Primers amplifying exon2 and exon3 to exon7 were used, respectively. Lcn6 expression of each mouse was first normalized to internal control (Gapdh), then relative Lcn6 mRNA level of Lcn6Δ/Δ mice was compared to that of control Lcn6fl/fl mice. Data are shown as the mean ± SEM, compared by unpaired Student t-test (two tailed). Each group consisted of 5 mice. ***P < 0.0001. (D) In situ hybridization analysis of Lcn6 mRNA level in the proximal caput of epididymides in Lcn6fl/fl and Lcn6Δ/Δ mice, respectively. Hybridization signal with the sense probe from the sample of Lcn6fl/fl mouse epididymis was used as negative control (N. C.). Scale bar = 50 μm. All the mice appeared normal and were unaffected by the introduction of loxP sites or TM treatment. When TM-treated mice were sacrificed, the weight of body, testes, and epididymides were recorded. No significant difference was found between Lcn6Δ/Δ male mice and the control Lcn6fl/fl male mice (see Supplementary Figure S3). As shown in Figure 4, histology of seminiferous tubules and epididymal structure in Lcn6Δ/Δ males did not show obvious abnormality. Tubules were filled with sperm and organization of epithelial cells in each segment of epididymides had no obvious changes. This suggests that LCN6 deficiency has no influence on the structure and organization of epididymal epithelium. Figure 4. View largeDownload slide Morphology of testis and epididymis in the Lcn6fl/fl and Lcn6Δ/Δ male mice. Images of left column showing the representative HE staining of testis and epididymis of caput, corpus, and cauda region in Lcn6fl/fl mice. Images of right column showing the representative HE staining of corresponding structure in Lcn6Δ/Δ mice. (A) and (E) Testis, (B) and (F) Caput epididymis, (C) and (G) Corpus epididymis, (D) and (H) Cauda epididymis. Regions in the dotted boxes are magnified in the bottom right corner. Scale bar (white) = 100 μm, scale bar (black) = 20 μm. Figure 4. View largeDownload slide Morphology of testis and epididymis in the Lcn6fl/fl and Lcn6Δ/Δ male mice. Images of left column showing the representative HE staining of testis and epididymis of caput, corpus, and cauda region in Lcn6fl/fl mice. Images of right column showing the representative HE staining of corresponding structure in Lcn6Δ/Δ mice. (A) and (E) Testis, (B) and (F) Caput epididymis, (C) and (G) Corpus epididymis, (D) and (H) Cauda epididymis. Regions in the dotted boxes are magnified in the bottom right corner. Scale bar (white) = 100 μm, scale bar (black) = 20 μm. Fertility test of conditional Lcn6 knockout mice Next, we performed mating test to examine natural fertility of Lcn6 null mice. During the 6-month mating, Lcn6fl/fl mating pairs gave birth to 22 liters (N = 3) while Lcn6Δ/Δ mating pairs produced 35 liters (N = 5). The number of average pups of per liter was 5.909 ± 0.4554 and 6.771 ± 0.4585, respectively. No difference was observed in the unstricted fertility test between Lcn6Δ/Δ mating pairs and Lcn6fl/fl mating pairs (Figure 5). This demonstrates that the loss of Lcn6 does not impair the fertility of male mice in vivo. Figure 5. View largeDownload slide Fertility test of Lcn6fl/fl and Lcn6Δ/Δ male mice. Numbers of pups per litter were obtained by crossing Lcn6fl/fl (N = 3) and Lcn6Δ/Δ (N = 5) male mice to normal WT female partners. Results are presented as the mean ± SEM, compared by Student t-test (two-tailed). Figure 5. View largeDownload slide Fertility test of Lcn6fl/fl and Lcn6Δ/Δ male mice. Numbers of pups per litter were obtained by crossing Lcn6fl/fl (N = 3) and Lcn6Δ/Δ (N = 5) male mice to normal WT female partners. Results are presented as the mean ± SEM, compared by Student t-test (two-tailed). Sperm functional analysis in conditional Lcn6 knockout mice Sperm in vitro fertilization ability, motility, and capacitation status Subsequently, we analyzed the cauda epididymal sperm from Lcn6Δ/Δ males in vitro. At first, sperm from Lcn6Δ/Δ males and control Lcn6fl/fl males were capacitated and subjected to in vitro fertilization. Six-hour incubation of sperm and cumulus-intact oocytes was first performed to evaluate the fertilization capability of Lcn6Δ/Δ sperm. In consequence, when adequate time was offered, Lcn6Δ/Δ sperm had the same ability as Lcn6fl/fl control sperm in fertilizing cumulus-intact eggs. We wondered whether long time compromised the dysfunction of Lcn6Δ/Δ sperm, thus 3-h incubation of sperm and cumulus-intact oocytes was implemented. However, Lcn6Δ/Δ group had the same 2-cell rate as the control group. These results demonstrate that Lcn6Δ/Δ sperm are as capable as Lcn6fl/fl sperm in fertilizing cumulus-intact eggs over a long time span (6 h) or a shorter time span (3 h) (Figure 6A). Figure 6. View largeDownload slide Unaltered sperm in vitro fertilization ability, motility, and capacitation status in Lcn6Δ/Δ mice. (A) 2-cell rates were counted 24 h after Lcn6fl/fl and Lcn6Δ/Δ cauda epididymal sperm fertilizing superovulated eggs. The rates of fertilization are shown, respectively, under incubation time of 3 h and 6 h with the eggs. All the results from independent experiments are plotted and presented as the mean ± SEM, compared by Student t-test (two-tailed). (B) Dynamic changes in motility parameters of Lcn6fl/fl and Lcn6Δ/Δ sperm during 120-min capacitation process. Each group consisted of 4 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed). VAP, average path velocity; VSL, straight-line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement. (C) Western blot analysis of protein tyrosine phosphorylation in Lcn6fl/fl and Lcn6Δ/Δ sperm during capacitation in the indicated time. The expression of α-tubulin was used as loading control. Figure 6. View largeDownload slide Unaltered sperm in vitro fertilization ability, motility, and capacitation status in Lcn6Δ/Δ mice. (A) 2-cell rates were counted 24 h after Lcn6fl/fl and Lcn6Δ/Δ cauda epididymal sperm fertilizing superovulated eggs. The rates of fertilization are shown, respectively, under incubation time of 3 h and 6 h with the eggs. All the results from independent experiments are plotted and presented as the mean ± SEM, compared by Student t-test (two-tailed). (B) Dynamic changes in motility parameters of Lcn6fl/fl and Lcn6Δ/Δ sperm during 120-min capacitation process. Each group consisted of 4 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed). VAP, average path velocity; VSL, straight-line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement. (C) Western blot analysis of protein tyrosine phosphorylation in Lcn6fl/fl and Lcn6Δ/Δ sperm during capacitation in the indicated time. The expression of α-tubulin was used as loading control. It is well known that ejaculated sperm have to swim through the female reproductive tract and capacitate there before fertilizing eggs in vivo. Thus, sperm motility and capacitation status are important parameters for fertilization potential. We assessed the motility of cauda epididymal sperm of Lcn6Δ/Δ and control mice by a computer-assisted sperm analysis system. During the whole 120-min process of capacitation in BWW medium, the total motility and progressive motility were still indistinguishable between control and Lcn6 null mouse sperm. Other motility parameters including average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) were slightly elevated in Lcn6Δ/Δ sperm through the capacitation process, though without significant statistical significance (Figure 6B). A distinguishing feature associated with sperm capacitation is the increase in protein tyrosine phosphorylation. Thus tyrosine phosphorylation of sperm proteins was monitored by Western blot during the capacitation process. We found Lcn6Δ/Δ sperm had the normal protein tyrosine phosphorylation showing gradual increase over time as the control sperm did (Figure 6C). Sperm acrosome reaction AR is important for sperm to fertilize the egg. Therefore, the ability of cauda epididymal sperm from Lcn6Δ/Δ males to undergo AR was assessed. When non-capacitated or capacitated for 1 h, Lcn6Δ/Δ sperm showed an enhanced spontaneous AR frequencies in comparison to control (P < 0.01) (Figure 7A; see representative pictures in Supplementary Figure S4). It suggests the deficiency of LCN6 protein leads to disordered spontaneous AR of sperm. To further investigate the capability of Lcn6Δ/Δ sperm in induced AR, capacitated sperm from the Lcn6Δ/Δ and control mice were, respectively, treated with Ca2+ ionophore A23187 and subjected to the AR frequency assessment. Nevertheless, no statistical difference was found between the Lcn6Δ/Δ sperm and the control (Figure 7A; see representative pictures in Supplementary Figure S4). It indicates Lcn6Δ/Δ sperm show the same response to A23187 as the control do. Figure 7. View largeDownload slide Impaired sperm spontaneous AR and intracellular calcium level in Lcn6Δ/Δ mice. (A) Comparison of sperm spontaneous and Ca2+ ionophore A23187 induced AR frequency between Lcn6fl/fl and Lcn6Δ/Δ male mice. Sperm spontaneous AR frequencies are, respectively, shown in the capacitation status (1 h) or not (0 h). Induced AR frequencies are obtained from sperm capacitated for 1 h. Each group consisted of at least 3 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed), **P < 0.01. (B) Comparison of intracellular calcium level of Lcn6fl/fl and Lcn6Δ/Δ sperm without capacitation (left panel) or under 1 h capacitation (right panel) in the indicated medium. BWW-Ca, BWW buffer without calcium. Each group consisted of 4 mice, data are shown as the mean ± SEM, compared by Student t-test (two-tailed). **P < 0.01. Figure 7. View largeDownload slide Impaired sperm spontaneous AR and intracellular calcium level in Lcn6Δ/Δ mice. (A) Comparison of sperm spontaneous and Ca2+ ionophore A23187 induced AR frequency between Lcn6fl/fl and Lcn6Δ/Δ male mice. Sperm spontaneous AR frequencies are, respectively, shown in the capacitation status (1 h) or not (0 h). Induced AR frequencies are obtained from sperm capacitated for 1 h. Each group consisted of at least 3 mice, data are presented as the mean ± SEM, compared by Student t-test (two-tailed), **P < 0.01. (B) Comparison of intracellular calcium level of Lcn6fl/fl and Lcn6Δ/Δ sperm without capacitation (left panel) or under 1 h capacitation (right panel) in the indicated medium. BWW-Ca, BWW buffer without calcium. Each group consisted of 4 mice, data are shown as the mean ± SEM, compared by Student t-test (two-tailed). **P < 0.01. Sperm AR is under precise control of numerous factors, of which calcium is the most important. Thus, we measured the intracellular calcium level of Lcn6 null sperm by using calcium indicator Fura2-AM. The intracellular calcium level in complete BWW medium was first analyzed. As shown in Figure 7B, whether sperm were capacitated for 1 h or not, Lcn6Δ/Δ sperm exhibited elevated intracellular calcium levels compared to control (P < 0.01). Then we wanted to know whether the increased calcium level of the Lcn6Δ/Δ sperm could be influenced by the extracellular calcium. Sperm from the Lcn6Δ/Δ and control mice were released into BWW medium without calcium, and then subjected to analysis of intracellular calcium level. Surprisingly, no difference can be detected when calcium was depleted from the external medium (Figure 7B). All these results suggest that LCN6 deficiency leads to impaired intracellular calcium balance of sperm. Discussion Previous studies from us and other groups have shown the involvement of lipocalins in sperm function in vitro [13–15,17]. However, these reports fail to elucidate their physiological roles in vivo. Our present study for the first time investigates and identifies the in vivo function of LCN6, an epididymis-specific lipocalin protein. It might be helpful to shed light on the physiological roles of epididymal lipocalins. As mentioned before, we suppose the generation of the animal model specifically deleting Lcn6 gene in adulthood might avoid possible complementary mechanism of molecular function during epididymal development. This advantage is supported by the unaltered expression of other lipocalins in epididymides of Lcn6 null mice (see Supplementary Figure S5). Nevertheless, the disadvantage of failing to reveal importance of individual gene when cluster of genes have some redundancy in their function is the same as that of other conventional knockout strategies. We speculate that the unchanged fertilization capacity of conditional Lcn6 knockout mice after Lcn6 deficiency (Figure 5) could be attributed to that. The epididymis-specific lipocalins are so important for male fertility, therefore functional substitution might arise during fertilization. This can somewhat be suggested by the existence of the epididymis-specific lipocalin cluster and their similar features of dependence on testicular factors and androgen [10,12,16,37]. From this point of view, it might be of great value to delete this cluster of lipocalins in the future to investigate their specific function in epididymis. However, we cannot exclude the possibility that mouse LCN6 might function in the development of epididymis since we only investigate the influence of LCN6 deficiency on adult mice. Our future work will focus on that. Though the unaltered male fertility of adult Lcn6 null mice, it is deserved to be mentioned that this mouse model is a meaningful attempt to targeted disruption of epididymis-specific gene in time-space manner. Several Cre recombinase transgenic mice that express the Cre recombinase under the control of epididymis-specific genes (e.g. Crisp4 [29], Defb41 [28], Lcn5 [38], and Rnase10 [39]) had been constructed to accomplish targeted elimination of genes. The benefits of these Cre mouse lines are obvious, so are the drawbacks. In these models, gene inactivation recombination event only occurs in particular regions at specialized time, of which most is before puberty. For example, the Cre recombinase expression of Crisp4-Cre, Defb41-Cre, and Rnase10-Cre mice started at Postnatal Day 20, and of the Lcn5-Cre mouse, it initiated at Day 30. Cre activity in all of these mouse lines is confined to the caput epididymis. Nevertheless, sperm maturation happens after sexual maturity and many genes specifically expressed in the corpus and cauda epididymis are actively involved in the maturation process. Therefore, conventional knockout strategies in epididymal research using existing Cre mouse lines are unlikely to meet the demand. In the present study, we accomplished a nearly complete deletion of Lcn6 in adult epididymis by using an extensively expressed and TM induced Cre line. And we can even delete Lcn6 gene in childhood of the mice if necessary. Furthermore, it is the first case that a TM induced knockout mouse model has been established in the epididymal research. During this process, we have not found any visible side effects on male fertility under this dosage of TM. But we are honest to say that TM is a kind of anti-estrogen drug and other researchers on male fertility should take this into consideration and observe its effects seriously when using it. We characterized two transcript variants of mouse Lcn6 that were specifically detected in the epididymis among all the tested mouse tissues. However, a sole transcript was found in the epididymis of human and rat [40]. Based on that, we wondered whether each of the mouse Lcn6 transcripts had its own function. Thus, we made a great effort on the production of anti-mouse LCN6α and LCN6β antibody. Unfortunately, all the antibodies, whether commercial or produced by ourself, failed to detect the in vivo LCN6 protein. Therefore, the respective function of mouse LCN6 isoforms is not able to be exposed. When orchidectomy and testosterone injections were performed in WT epididymis, the changes in the mRNA level of Lcn6 suggest its regulation by androgen. But its expression not getting complete recovery after testosterone replacement (Figure 2) indicates that other factors in testicular fluid are involved. It has long been proved that testosterone levels restoration could reverse changes in the caput, corpus, and cauda epididymis after orchidectomy, but not in the initial segment [41]. This is in accordance with the fact that Lcn6 mRNA exists mostly in the initial segment and some in the other parts of caput epididymis (Figure 1). We speculate that LCN6 protein is regulated by testicular factors as well as androgen and it possesses important functions in the epididymis. When both transcripts are null in Lcn6 knockout mice, no obvious change could be observed in the morphology of epididymides (Figure 4), while cauda epididymal sperm from the Lcn6 knockout mice indeed have a premature AR (Figure 7). This demonstrates the function of LCN6 on regulation of sperm AR. Furthermore, our in vitro experimental results show that only mLCN6α is inclined to be secreted into extracellular environment when 3× flag-tagged mLCN6α and mLCN6β protein were over-expressed in HEK293T cell, respectively or simultaneously. The flag fusion mLCN6α protein even can be incorporated onto/into Lcn6 deficient sperm head, exactly between the equatorial segment and the post-acrosomal region (see Supplementary Figure S6). These indicate that LCN6 protein, especially LCN6α, might help to prevent the happening of premature AR. To fuse with oocytes, mammalian sperm must pass through the cumulus cell layer and the zona pellucida (ZP) of eggs. At this point, the AR is initiated [42–44]. The acrosomal reaction normally takes place in the present of ovum. But a certain percentage of sperm undergo spontaneous AR without the presence of oocytes [45]. This fundamental maturational process is regulated by numerous signaling cascades, and calcium plays a dynamic role in this process [46,47]. It is known that a high and sustained calcium influx is necessary for the AR to occur and Ca2+ ionophore that elevates intracellular calcium level can increase the AR frequency [48–50]. In fact, the ability to undergo Ca2+ ionophore A23187 induced AR could be used as a clinical marker to evaluate the fertilization potential of sperm [51–53]. In Lcn6 deficient sperm, spontaneous AR frequency is higher than control sperm whether capacitated or not, but Ca2+ ionophore A23187 induced AR frequency has no significant change (Figure 7). These evidences imply that LCN6 deficiency leads to impaired sperm maturation in AR to a certain extent, without harm to the fertilization potential of the sperm. However, the elevation of spontaneous AR frequency is in accompany with the increase of intracellular calcium level in Lcn6 knockout sperm, and that rise of intracellular calcium level depends on the calcium in the medium (Figure 7). This is consistent with the fact that spontaneous AR is strictly dependent on the presence of extracellular calcium [54]. These evidences indicate that LCN6 deficiency leads to the rise of calcium influx in the sperm and possibly LCN6 protein is involved in the regulation of calcium influx. Cauda epididymal sperm from the Lcn6 null mouse still gain the ability to fertilize the egg in vitro (Figure 6). Though this is in conformity with their phenotype of nonimpaired Ca2+ ionophore A23187 induced AR frequency, it is generally considered that sperm in the early stages of AR may bind to and go on to penetrate the ZP, but those that undergo AR prematurely will be severely compromised in their ability to fertilize [55]. As a matter of fact, Jin et al. investigated the mouse sperm AR status before and during the fertilizing process, it turned out that most fertilizing sperm underwent the AR before reaching the ZP of cumulus-enclosed oocytes [56]. Inoue et al. even demonstrated the possibility of that acrosome-reacted mouse sperm recovered from the perivitelline space can fertilize other eggs [57]. Based on these evidence, we consider our investigation on Lcn6 deficient sperm provides further evidence for the theory of that premature AR in a certain range is nonlethal to the in vitro fertilization potential of sperm. When it comes to enhanced spontaneous AR’s impact on in vivo fertilization, it seems less optimistic. Considerable references show that ejaculated sperm have a basic low level of spontaneous AR rate until they get close enough to the ovulated eggs [58–60]. That means an impaired fecundity should be observed in Lcn6 null mice when their sperm exhibit premature AR. But in fact, it is not the case. In addition to the above mentioned possible functional redundancy of lipocalins, we speculate this phenomenon could be interpreted from the other perspective. Functional substitution is not always limited to the same gene family although in most case it is [61,62]. In fact, CD46 deficient mice exhibited similar phenotype as the Lcn6 null mice, which had an accelerated spontaneous AR of sperm and increased male fertility [63]. That indicates the significance of sperm-oocyte interaction and existence of cumulatively functioning genes in the course. So there is still a possibility that non-lipocalins might substitute for the lost of LCN6 function, which could give rise to the unaltered fecundity of Lcn6 null mice. In summary, we conclude that mouse Lcn6 is not required for male fecundity, but has an irreplaceable role in sperm maturation. Supplementary data Supplementary data are available at BIOLRE online. Supplemental information contains Supplemental Experimental Procedures, six figures, and one table that are available online. Supplementary Figure S1. The location of Lcn6 gene on mouse chromosome, with the arrangement of other lipocalin genes in the gene cluster. Supplementary Figure S2. The cDNA and amino acid sequence of two isoforms of mLcn6. Supplementary Figure S3. (A) Photography of testes and epididymides of Lcn6fl/fl and Lcn6Δ/Δ male mice. (B) Comparison of the ratio of testis/body weight and epididymis/body weight between Lcn6fl/fl and Lcn6Δ/Δ male mice. Supplementary Figure S4. Representative pictures in assessment of spontaneous AR frequency and Ca2+ ionophore A23187 induced AR frequency in Lcn6 null mice and control Lcn6fl/fl mice. Supplementary Figure S5. qRT-PCR analysis of mRNA level of other lipocalin genes in Lcn6 null mice and control Lcn6fl/fl mice. Supplementary Figure S6. Expression of 3× flag-tagged mLCN6α protein and its incorporation onto/into Lcn6 deficient sperm. Supplementary Table S1. Primers that were used in the manuscript. Author Contributions: Jia Shen prepared Figures 1 and 2 and Qianqian Yin prepared Figures 3–7. Xiaofeng Wang constructed the animal model. Qianqian Yin wrote the main manuscript text, Yonglian Zhang, Qiang Liu and Yuchuan Zhou gave the guidance and revised the paper. All authors reviewed the manuscript. 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Biology of ReproductionOxford University Press

Published: Jan 1, 2018

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