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. 2011 Jan;84(1):140-52.
doi: 10.1095/biolreprod.110.084855. Epub 2010 Sep 1.

Reduced fertility in vitro in mice lacking the cystatin CRES (cystatin-related epididymal spermatogenic): rescue by exposure of spermatozoa to dibutyryl cAMP and isobutylmethylxanthine

Kim M Chau  1 , Gail A Cornwall
Affiliations

Reduced fertility in vitro in mice lacking the cystatin CRES (cystatin-related epididymal spermatogenic): rescue by exposure of spermatozoa to dibutyryl cAMP and isobutylmethylxanthine

Kim M Chau et al. Biol Reprod. 2011 Jan.

Abstract

The cystatin CRES (cystatin-related epididymal spermatogenic; Cst8) is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors and is present in the epididymis and spermatozoa, suggesting roles in sperm maturation and fertilization. To elucidate the role of CRES in reproduction, mice lacking the Cst8 gene were generated and their fertility examined. Although both male and female Cst8(-/-) mice generated offspring in vivo, spermatozoa from Cst8(-/-) mice exhibited a profound fertility defect in vitro. Compared to spermatozoa from Cst8(+/+) mice, spermatozoa from Cst8(-/-) mice were unable to undergo a progesterone-stimulated acrosome reaction and had decreased levels of protein tyrosine phosphorylation, suggesting a defect in the ability of Cst8(-/-) spermatozoa to capacitate. Incubation of Cst8(-/-) spermatozoa with dibutyryl cAMP and 3-isobutyl-1-methylxanthine rescued the fertility defect, including the capacity for sperm protein tyrosine phosphorylation. Both untreated Cst8(+/+) and Cst8(-/-) spermatozoa, however, exhibited similar increased total levels of cAMP and protein kinase A (PKA) activity throughout the capacitation time course compared to spermatozoa incubated under noncapacitating conditions. Taken together, these results suggest that mice lacking CRES may have altered local levels of cAMP/PKA activity, perhaps because of improper partitioning or tethering of these signaling molecules, or that the CRES defect does not directly involve cAMP/PKA but other signaling pathways that regulate protein tyrosine phosphorylation and capacitation.

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Figures

FIG. 1.
FIG. 1.
Production of mice lacking Cst8. A) _targeting construct. The first two Cst8 exons (vertical boxes) were replaced by neo. Restriction enzyme sites: B, BamHI; Bsi, BsiWI; H, HindIII; RI, EcoRI; RV, EcoRV; X, XhoI. WT2, N1, and SA5 are primers used for PCR genotyping of the WT (WT2 and SA5) and KO (N1 and SA5) alleles. B) PCR analysis of genomic DNA isolated from tail snips from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice for the Cst8 mutation. C) Northern blot analysis of CRES mRNA levels in testis (TE) and epididymal (EPI) tissue isolated from Cst8+/+, Cst8+/−, and Cst8−/− mice and probed with a radiolabeled CRES cDNA. The blot was stripped and reprobed with a cDNA generated against 18S rRNA to confirm equal loading. D) Western blot analysis of CRES protein in tissue lysates prepared from testis (TE) and epididymis (EPI) from Cst8+/+, Cst8+/−, and Cst8−/− mice.
FIG. 2.
FIG. 2.
Analysis of Cst8+/+ and Cst8−/− sperm fertilizing ability in vitro. A) Percentage of COCs fertilized after 3 h of incubation with control CD1, Cst8+/+, and Cst8−/− spermatozoa (n = 5; a vs. b, P ≤ 0.05; a vs. c, P ≤ 0.001; b vs. c, P ≤ 0.01). B) Number of Cst8+/+ and Cst8−/− spermatozoa bound to the zona pellucida of cumulus-free oocytes from CD1 female mice. Spermatozoa were also incubated with 2-cell embryos as a control for nonspecific binding (n = 4; a vs. b, P ≤ 0.01; a vs. c, P ≤ 0.001; b vs. c, P ≤ 0.05). C) Percentage sperm-egg fusion of cumulus- and zona pellucida-free oocytes from CD1 females incubated with spermatozoa from control CD1, Cst8+/+, and Cst8−/− mice (n = 5; a vs. b, P ≤ 0.05; a vs. c, P ≤ 0.001; b vs. c, P ≤ 0.01). Values represent the mean ± SEM.
FIG. 3.
FIG. 3.
Sperm motility in Cst8+/+ and Cst8−/− mice. Equal concentrations of spermatozoa from Cst8+/+ (black bars) and Cst8−/− (white bars) mice were examined after varying times of capacitation. In each experiment, 10 fields were examined per sample. Values represent the mean ± SEM of four experiments. % motile, percentage of spermatozoa with motility; % progressive motility, percentage of spermatozoa moving forward. An asterisk indicates statistical significance (*P < 0.05).
FIG. 4.
FIG. 4.
Sperm capacitation and acrosome reaction in Cst8+/+ and Cst8−/− mice. A) Spermatozoa from Cst8+/+ (filled circles) and Cst8−/− (filled squares) mice after various times of capacitation were induced to undergo the acrosome reaction by the calcium ionophore A23187 (left) or progesterone (right). Spermatozoa from Cst8+/+ (open circles) and Cst8−/− mice (open squares) that were incubated in DMSO vehicle only and, thus, represent spontaneous acrosome reactions are also shown. The present or absence of the acrosome was determined by Coomassie Blue staining. Values represent the mean ± SEM (A23187, n = 3; progesterone, n = 4). An asterisk indicates statistical significant (*P ≤ 0.001) comparing Cst8+/+ to Cst8−/− spermatozoa in the presence of progesterone. B) Western blot analysis of protein tyrosine phosphorylation in Cst8+/+ and Cst8−/− spermatozoa after increasing periods of time in capacitation buffer. The blot is representative of that from three different sperm capacitation experiments. Coomassie Blue staining of the blot confirmed equal loading of protein in each lane (not shown).
FIG. 5.
FIG. 5.
Rescue of sperm protein tyrosine phosphorylation and fertility in spermatozoa from Cst8−/− mice after incubation in dbcAMP and IBMX. A) Western blot analysis of protein tyrosine phosphorylation in Cst8+/+ and Cst8−/− spermatozoa after increasing periods of time in capacitation buffer and in the presence of dbcAMP and IBMX or the vehicle DMSO. Blots were stripped and reprobed with anti-tubulin antibody to verify equal loading of the gel. Blots are representative of those from three different experiments. B) Percentage of COCs fertilized after 3 h of incubation with Cst8+/+ and Cst8−/− spermatozoa that were untreated, incubated with dbcAMP, or incubated with dbcAMP and IBMX before addition to the oocytes. Values represent the mean ± SEM (n = 10 experiments; a vs. b, P ≤ 0.05; c vs. a, P ≤ 0.001; c vs. b, P ≤ 0.05; c vs. ab, P ≤ 0.01; bc vs. a, P ≤ 0.01).
FIG. 6.
FIG. 6.
Cyclic AMP levels in Cst8+/+ and Cst8−/− spermatozoa. Cell extracts prepared from Cst8+/+ (circles) and Cst8−/− (squares) spermatozoa incubated in capacitating (filled symbols) or noncapacitating (open symbols) media were analyzed for total levels of cAMP after various times of capacitation. Values represent the mean ± SEM (n = 10 experiments). Cyclic AMP levels in Cst8+/+ and Cst8−/− spermatozoa were significantly increased at all time points in spermatozoa incubated in capacitating medium compared to spermatozoa incubated in noncapacitating medium.
FIG. 7.
FIG. 7.
PKA activity in Cst8+/+ and Cst8−/− spermatozoa during capacitation. A) PKA activity in Cst8+/+ (circles) and Cst8−/− (squares) spermatozoa after incubation in capacitating (filled symbols) or noncapacitating (open symbols) medium. Values represent the mean ± SEM (n = 11 experiments). An asterisk indicates statistical significance (*P ≤ 0.05) comparing Cst8+/+ spermatozoa to Cst8−/− spermatozoa. The PKA activity in Cst8+/+ and Cst8−/− spermatozoa incubated under capacitating conditions was increased at all time points compared to PKA activity of spermatozoa incubated under noncapacitating conditions (P ≤ 0.05). PKA activity in capacitated Cst8+/+ spermatozoa was not different from that in capacitated Cst8−/− spermatozoa except at 90 min (P ≤ 0.05). B) PKA activity was measured in similar samples as in A but with 10 μM H89, a PKA inhibitor, included during the assay (n = 2 experiments). C) PKA activity was measured in similar samples as in A but with 1 mM dbcAMP and 100 μM IBMX included during the assay (n = 9 experiments).
FIG. 8.
FIG. 8.
Phosphotyrosine fluorescence in capacitated Cst8+/+ and Cst8−/− spermatozoa. A) At 0 and 90 min of capacitation, Cst8+/+ (WT) and Cst8−/− (KO) spermatozoa were air-dried onto slides and incubated with the monoclonal anti-phosphotyrosine antibody followed by an Alexa Fluor 594-labeled secondary antibody. All images were captured using the same exposure times and were set to that of the strongest fluorescent labeling in WT 90-min sperm samples. Fluorescent images are superimposed on those obtained under phase contrast. Original magnification ×40. B) Percentage of spermatozoa that exhibited phosphotyrosine fluorescence on the midpiece and principle piece only (tail), tail plus full head fluorescence (full), or tail and partial head fluorescence (partial) at 0 and 90 min of capacitation. Value represent the mean ± SEM (n = 3 experiments).
FIG. 9.
FIG. 9.
Phosphotyrosine fluorescence patterns observed on the sperm heads of Cst8+/+ and Cst8−/− spermatozoa. Images are to show pattern only and are not relative fluorescence levels between the varying patterns. A) Full fluorescence over most of the sperm head; partial head staining included that in the triangular, acrosomal, or other patterns. Fluorescent images are superimposed on those obtained under phase contrast. Original magnification ×40. B) Percentage of Cst8+/+ (WT) and Cst8−/− (KO) sperm heads exhibiting the triangular, acrosomal, or other patterns of phosphotyrosine fluorescence after 90 min of capacitation. Value represent the mean ± SEM (n = 3 experiments). C) Percentage of WT and KO sperm heads exhibiting triangular (tri), acrosomal (acr), or other patterns of phosphotyrosine fluorescence after 90 min of capacitation in the presence of DMSO or 1 mM dbcAMP plus 100 μM IBMX. Value represent the mean ± SEM (n = 4 experiments). An asterisk indicates statistical significance (*P < 0.05).
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References

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