Survival of mouse type B spermatogonia for the study of the biological effectiveness of 1 MeV, 2.3 MeV and 5.6 MeV fast neutrons.

The most radiation-sensitive cells in the testis are B and intermediate spermatogonia. We have used a histological scoring technique to compare three neutron beams of different mean energies (1 MeV at the ECN, Petten, 2.3 MeV at the Gray Laboratory, Northwood, and 5.6 MeV at the Oncological Centre, Krakow). CBA inbred mice, 14-20 weeks old, were exposed to whole-body irradiation with single doses of either X-rays (0.1-1 Gy) or neutrons (0.2-0.25 Gy). Relative biological effectiveness values, calculated at the level of 50 per cent reduction in survival of B spermatogonia, were 5.7 at the ECN, Petten, 4.6 at the Gray Laboratory and 3.0 at the Oncological Centre in Krakow. The Do value for the B spermatogonia after X-rays was 0.34 +/- 0.02 Gy when the data from the three centres were combined. Do values for neutrons for the examined spermatogonia were 0.08 Gy, 0.09 Gy and 0.11 Gy at the ECN, Petten, the Gray Laboratory and the Oncological Centre, respectively.

Reversible harmless interruption of testicular blood supply in the ram.

An effective method of interrupting testicular blood flow temporarily and repeatedly in the ram has been developed. Blockade of flow has been achieved mechanically by an inflatable occluder placed around the testicular artery at the level of the spermatic cord. The effect of the blockade on total testicular blood supply was investigated using Doppler flowmetry and a percutaneous Xenon-133 injection method. With both approaches, the blood flow changes after inflation or deflation of the occluders could be estimated satisfactorily. A substantial decrease of testicular blood flow was achieved in eight of the 10 testes with inflated occluders. However, there were indications that in the remaining two testes blockade of the arterial flow was not complete. After deflation of the occluders, blood flow was restored rapidly and completely in all testes. Macro- and microscopic examinations revealed no long-term damage to the testis after blood flow interruptions lasting 30 or 60 minutes.

Cell-cell interaction between rat Sertoli cells and mouse germ cells in vitro.

An antiserum against rat germ cell membranes was prepared, and after absorption with protein extracts of rat liver and kidney and mouse testis, this antiserum reacted only with rat germ cell membranes and juxtanuclear vesicles in rat Sertoli cells. Germ cell-free rat Sertoli cell monolayers were cultured in vitro. Freshly isolated mouse germ cells adhered to these monolayers within 1 h. After a minimum of 3 days of such a co-culture, immunofluorescence and immunoblotting revealed that the mouse germ cells had obtained rat-antigenic determinants in their membranes. Our results indicate that this appearance of rat-specific antigens on mouse germ cells is specific and inducible.

Strain differences in the response of mouse testicular stem cells to fractionated radiation.

The survival of spermatogonial stem cells in CBA and C3H mice after single and split-dose (24-hr interval) irradiation with fission neutrons and gamma rays was compared. The first doses of the fractionated regimes were either 150 rad (neutrons) or 600 rad (gamma). For both strains the neutron survival curves were exponential. The D0 value of stem cells in CBA decreased from 83 to 25 rad upon fractionation; that of C3H stem cells decreased only from 54 to 36 rad. The survival curves for gamma irradiation, which all showed shoulders, indicated that C3H stem cells had larger repair capacities than CBA stem cells. However, the most striking difference between the two strains in response to gamma radiation was in the slopes of the second-dose curves. Whereas C3H stem cells showed a small increase of the D0 upon fractionation (from 196 to 218 rad), CBA stem cells showed a marked decrease (from 243 to 148 rad). The decreases in D0 upon fractionation, observed in both strains with neutron irradiation and also with gamma irradiation in CBA, are most likely the result of recruitment or progression of radioresistant survivors to a more sensitive state of proliferation or cell cycle phase. It may be that the surviving stem cells in C3H mice are recruited less rapidly and synchronously into active cycle than in CBA mice. Thus, it appears that the strain differences may be quantitative, rather than qualitative.

Growth and differentiation of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons.

The longitudinal outgrowth of spermatogenetic colonies arising from stem cells that survived neutron doses of 150, 300, and 350 rad was studied up to 30 weeks in histological sections of CBA mouse testes. Two methods were used: (1) the repopulation index (RI) as a measure of the length of total colonies per testis and (2) measurement of the individual length of all colonies in serially sectioned testes 4 and 15 weeks after 300 rad and 15 weeks after 350 rad. The mean initial growth of the colonies is linear up to 8, 15, and 20 weeks after 150, 300, and 350 rad, respectively. Although after these doses the mean initial colony growth rate did not differ significantly (about 27 microns/day), both methods showed that the colonies grow about 20% slower after 350 rad. Screening of individual colonies revealed a great variation in colony length per testis and a higher frequency of short colonies with higher neutron doses. Counting of colonies after 300 rad showed that all surviving stem cells had started to form a colony within 4 weeks after irradiation. The development of spermatogenetic cells to mature spermatozoa was studied after 100, 150, 300, and 350 rad in sections of repopulating tubules used for RI determination as well as in serial sections of individual colonies. Although after 300 and 350 rad spermatogenetic cell types beyond the stage of young spermatocytes reappeared 1 week late, we found no great disturbances in the regular reappearance of the successive spermatogenetic cell types after irradiation. However, from the study of individual colonies it appeared that colonies differ widely in their development even within one testis. Moreover, the frequency of less developed colonies was higher after 350 rad than after 300 rad. Our data suggest that this retardation in the reappearance of further developed cells is caused by a delay in the production of developed cells in spermatogonia in an increasing fraction of the colonies after higher neutron doses. Even in fully developed colonies the production of differentiating spermatogenetic cell types was subnormal after 300 and 350 rad. This was caused by an extensive cell degeneration in the colonies as well as by a tendency of the undifferentiated and/or A1-spermatogonial population to increase its own number at the cost of the production of further developed cells.