By contrast, although some seminiferous tubules in EDS-exposed mice appeared normal, other tubule cross-sections continued to have incomplete spermatogenesis with Sertoli cell only tubules.
Morphometric analysis revealed a decrease in the seminiferous tubule area, 57 516 ± 535 vs. 52 835 ± 1203.56 |xm2, an increase in the corresponding interstitial area, 7728 ± 535 vs. 12 409 ± 1204 |xm2. The smaller apparent tubule size in EDS-exposed testes coupled with a concomitant increase in interstitial space resulted in a perceptional increase in LC number. This apparent increase was analyzed using stereology to determine whether there were real alterations in the size of the LC population. Immunocyto-chemistry for P450scc enzyme was used to positively identify LCs in paraffin sections of adult testes (PND 52). Ste-reologic analysis of these tissue sections indicated that the total number of LC per testis was actually decreased in the EDS-exposed males, 5.7 X 106 ± 4.6 X 105 vs. 4.2 X 106 ± 2.8 X 105 LCs for control and EDS, respectively (Fig. 6). Analysis also revealed a decrease in the average volume of LCs, 1325.4 ± 96.9 vs. 779.6 ± 38.2 |xm3 as well as an increase in the LC volume density, 6.2% vs. 9.3%.
The average T production per testis from ex vivo incubations was divided by the average number of LCs per testis to estimate T production per LC. On either an unstimulated or hCG-stimulated basis, estimated T production per LC was increased in EDS-exposed males. The estimate for unstimulated T production was 9.3 X 10_9 ng T per LC and 373.4 X 10_9 ng T per LC for control and eDs, respectively. The estimate for stimulated T productions was 424.0 X 10 9 ng T per LC and 1979.0 X 10 9 ng T per LC for control and EDS, respectively.
Adult male fertility was assessed for differences in mating ratio (the number of total observed copulatory plugs divided by the number of female mice that cohabited with each male), the fertility ratio (the number of actual litters divided by the number of observed copulatory plugs), and litter size (Fig. 7). The mating ratio was reduced, 0.74 ± 0.04 vs. 0.56 ± 0.08, as was the fertility ratio, 0.96 ± 0.08 vs. 0.56 ± 0.13, and the size of the F2 litters, 12.7 ± 0.4 vs. 9.4 ± 1.3 pups.
FIG. 5. Light micrographs of testes from vehicle and EDS-exposed males. A) Cross section of the testis of a vehicle-exposed neonatal (PND 1) male. The germ cell layers have not yet formed and the LCs are dispersed uniformly throughout the inter-stitium. B) Testis from an EDS-exposed neonatal male. There are multiple areas of LC aggregates throughout the interstitium (arrow). C) Testis of a vehicle-exposed prepubertal (PND 24) male. Multiple germ cell layers have been formed and the lumen is visible. D) Testis from an EDS-exposed prepubertal mouse. The germ cell layers have not yet developed and the majority of tubule cross sections are Sertoli cell only (arrow). E) Testis from a vehicle-exposed adult (PND 82) male mouse. Spermatogenesis is complete with the complete development of the germ cell layers. F) Testis from an EDS-exposed adult male mouse. Spermatogenesis is still incomplete as evidenced by few normal germ cell associations and abundant Sertoli cell cytoplasm with vacant spaces or vacuoles (arrow). It appears that the interstitial space is larger and that LCs are more abundant.
FIG. 6. Light micrographs of paraffin-embedded, P450scc-stained, 4- to 6-^m cross sections of vehicle- and EDS-exposed testes. A) Cross section of a vehicle-exposed testis with P450scc enzyme immunostaining to positively identify LCs; brown reaction product indicates LC staining. B) Testis from an EDS-exposed male. Spermatogenesis is incomplete, as evidenced by the paucity of germ cell associations and there is an abundance of small, stained LCs within the interstitium. C) Summary of the stereology of testes from vehicle and EDS-exposed male mice. Values represent litter means ± SEM; statistical significance (P < 0.05 = * and P < 0.001 = **) is indicated; N = 9 and 10 for vehicle- and EDS-exposed groups, respectively. Estimates of T production per LC are also provided.
FIG. 7. Assessment of adult (PND 70-80) male fertility. Values represent litter means ± SEM; statistical significance (P < 0.05 = *) is indicated; N = 16 and 17 for vehicle- and EDS-exposed groups, respectively.