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1.
Fig 5

Fig 5. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Tissue Histology and Sperm Cytology. Hematoxylin-eosin stained section of testes (A, B) and epididymides (C, D), and representative phase contrast images of caudal epididymal sperm from four different Tpst2+/+ and Tpst2-/- mice (E and F) are shown.

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
2.
Fig 2

Fig 2. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Sulfotyrosine Analysis. Tissue explants were metabolically labeled with Na35SO4 and quantitative sulfotyrosine analysis was performed as described in Experimental Procedures. Sulfotyrosine analysis of secreted (A) and cellular proteins (B) from wild type (open bars), Tpst1-/-(gray bars), and Tpst2-/-(black bars) tissues are shown. Results are expressed as the mean ± SEM of three independent experiments.

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
3.
Fig 9

Fig 9. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Fertilization of ZP-free eggs. ZP-free eggs were prepared from CF-1 mice and inseminated with sperm from either Tpst2+/+ or Tpst2-/- mice for 60 min, then washed, fixed, and assessed for sperm-egg binding (A and C) or sperm-egg fusion (B and D). ZP-free eggs were inseminated with sperm at a sperm:egg ratio of 25:1 (A and B) or at a sperm:egg ratio of 100:1 (C and D). Results are expressed as mean ± SE from analyses of sperm from three wild type and four Tpst2-/- mice.

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
4.
Fig 4

Fig 4. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Reproductive Performance. Ten week-old virgin Tpst2+/- or Tpst2-/- females were mated with either Tpst2+/- or Tpst2-/- males. Females were examined for copulatory plugs each morning to determine the percentage of females plugged (A), the plugging latency (B), and the size of any firstborn litter (C). Results are expressed as the mean ± SD. The sample size is indicated in parentheses. Neither the percentages of females plugged (p = 0.478, two-tailed Fisher’s exact test) nor the plugging latencies (p = 0.072, two-tailed Student’s t-test with unequal sample variance) were statistically different between groups.

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
5.
Fig 8

Fig 8. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Sperm Capacitation and Acrosomal Exocytosis. A. The ability of sperm to capacitate in vitro as a function of time under capacitating (M199, 0.4% BSA, closed circles) or non-capacitating (M199 alone, open circles) conditions was assessed by fluorescence microscopy of sperm stained with FITC-CTß as described in Experimental Procedures. Results are expressed as mean ± SD of three independent experiments. B. The ability of sperm to undergo acrosomal exocytosis in response to 50 μM calcium ionophone A23187 (solid bars) or diluent (open bars) was assessed by fluorescence staining FITC-Peanut agglutinin as described in Experimental Procedures. Results are expressed as mean ± SD of four independent experiments.

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
6.
Fig 3

Fig 3. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Growth Curves. Offspring from heterozygote mating pairs were weighed weekly starting at 3 weeks of age and pups were weaned at 3 weeks. Results are shown as the age vs. mean ± SE of body weight in grams. The number of mice in each group were; Tpst2+/+ male (n = 39), Tpst2-/- male (n = 26), Tpst2+/+ female (n = 41), Tpst2-/- female (n = 34). Statistical significance between groups at each age was determined using a one-tailed Student’s t-test with unequal sample variance. (*p <0.05, **p = < 0.01, † p = <10-3, ‡ p = <10-4, § p = <10-5, §§ p = <10-7).

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
7.
Fig 6

Fig 6. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Analysis of Natural Matings. Each of ten virgin 10-12 week-old Tpst2+/+ and Tpst2-/- males was mated overnight with a superovulated FVB/N female. One week, and then again two weeks later, each male was mated overnight with a different superovulated female, for a total of two or three matings for each male. The morning after each mating, females were examined for copulatory plugs (A) and then euthanized. Egg clutches were collected from the ampullae of the oviducts, all the eggs collected were scored for fertilization (B), and the total number of eggs harvested from each female determined (C) as described in Experimental Procedures. Results are expressed as the mean ± SD. The sample size is indicated in parentheses. The difference in the percentage of eggs fertilized between groups was highlysignificant (p <10-32, two-tailed Fisher’s exact test). The number of eggs harvested per female was not statistically different (p = 0.773, two-tailed Student’s t-test with unequal sample variance).

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
8.
Fig. 1

Fig. 1. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Targeted Disruption of the Tpst2 Gene. A. Diagram of the mouse TPST-2 mRNA and corresponding primary structural features of the TPST-2 protein. The untranslated and translated portions of exon 3 are indicated by open and solid rectangles, respectively. The transmembrane domain is indicated by an open rectangle, N-glycosylation sites are indicated (lancet), and the location of the 5’ PSB and 3’ PB motifs common to the sulfotransferase gene family are shown. B. Diagram of the wild type Tpst2 locus, targeting vector, and targeted locus. A 0.88 kb fragment spanning the start codon and the remainder of exon 3 was replaced by a 1.7 kb PGKneo cassette. Triangles indicate the location of loxP sites. C. Southern analysis of XbaI digested genomic DNA. Homologous recombination at the Tpst2 locus adds an XbaI site that results in a shorter XbaI genomic fragment (2 kb) than that from the wild type allele (6 kb) that is detected by hybridization with Probe A. D. Northern analysis of total RNA from tissues of Tpst2+/+, Tpst2+/-, Tpst2-/- mice using either a TPST-2 (top), TPST-1 (middle), or control CHO-B (bottom) cDNA probe.

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.
9.
Fig 7

Fig 7. From: Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility.

Sperm Motility in Viscous Media. Swim-up fractions of sperm from wild type and Tpst2-/- males were prepared and capacitated as described in Experimental Procedures. Sperm were observed in normal viscosity media (M199-BSA) or in viscous media (2% long chain polyacrylamide in M199-BSA). A. Video images were analyzed off-line to determine the number of sperm that were inactive (immotile), active with no forward velocity (non-progressive), or active with forward velocity (progressive). Results are expressed as mean ± SD of the fraction of immotile, non-progressive, and progressive sperm from three different animals. The mean number of spermatozoa analyzed (mean ± SD, n = 3) were; Tpst2+/+ (n = 146 ± 33) and Tpst2-/-(n = 91 ± 30) in normal media, and Tpst2+/+ (n = 83 ± 13) and Tpst2-/-(n = 84 ± 6) in viscous media. B. Sperm velocities were measured by tracking individual cells frame-by-frame using NanoTrack Software (Isee Imaging Systems). Results are expressed as the mean ± SD of straight line velocities of progressively motile sperm in viscous media. The combined number of spermatozoa analyzed from the three different animals were; Tpst2+/+ (n = 62) and Tpst2-/-(n = 61). The difference in sperm straight line velocities was highly significant (p <10-23, two-tailed Student’s t-test with unequal sample variance).

Atefeh Borghei, et al. J Biol Chem. ;281(14):9423-9431.

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