Effects of a single brief period of moderate heating of the testes on seminiferous tubules in hypophysectomized rams treated with pituitary extract M. T. Hochereau-de Reviers, A. Locatelli, C. Perreau, C. Pisselet and B. P. Setchell Reproductive Physiology Station, INRA. URA CNRS 1291, Nouzilly 37380, Monnaie, France; and 2On study leave from Department of Animal Sciences, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond 5064, South Australia, Australia An experiment was conducted to examine the appearance of the seminiferous tubule 20 days after a single exposure of the testes of rams to a scrotal temperature of about 42\s=deg\Cfor 45 min. Ten of the animals were surgically hypophysectomized and five were simultaneously heated; these rams were treated twice a day with ovine pituitary extract to avoid modifi- cations in the negative feedback from the testes to the pituitary and consequent changes in gonadotrophin secretion. Six intact rams (three heated and three unheated) were also studied. The pituitary extract significantly increased the testis weight and spermatogonial multipli- cations from A1 spermatogonia onwards. Twenty days after the heat treatment, testis weight was significantly reduced by heating; both tubular and intertubular tissues were affected. The total length of seminiferous tubules per testis was not modified, whereas the mean seminiferous tubule diameter was significantly reduced after heating. The total number of Sertoli cells per testis was not significantly modified, while their mean cross-sectional nuclear area was significantly reduced by heat treatment. A decrease in the number of all germ cells except A0 spermatogonia, from A1 spermatogonia onwards, was observed. The number of round spermatids decreased by 95 and 90%, slightly more than the diplotene primary spermatocytes (76 and 77%) and elongated spermatids (79 and 85%) in hypophysectomized pituitary extract-treated and intact rams, respectively. Round and elongated spermatids would be derived from germ cells that were respectively leptotene and young pachytene primary spermatocytes at the time of heating, whereas diplotene primary spermatocytes would have been type B spermatogonia. Normal spermiation did not take place and some testicular spermatozoa remained loosely attached to the seminiferous epithelium. It is con- cluded that this mild heat treatment caused considerable disruption to the spermatogenic cells, but the effect was comparable in intact rams, in which pituitary gonadotrophin secretion may have changed and in hypophysectomized rams treated with a constant dose of pituitary extract. Introduction of heating on local paracrine control from effects due to changes in gonadotrophin secretion. Interstitial tissue volumes, semin¬ iferous tubule Sertoli and cell Degeneration of the seminiferous epithelium induced by a short parameters, germ populations period of local hyperthermia has been extensively studied since were analysed. the results obtained in rats by Steinberger and Dixon (1959), and Chowdhury and Steinberger (1964) and Collins Lacy (1969) Materials and Methods and in rams by Waites and Ortavant (1968). However, after that induce of modifi¬ treatments disruption spermatogenesis, Animals cations of the intratesticular local paracrine control cannot be in secretion which dissociated from changes gonadotrophin Sixteen Ile de France adult rams were used during the season could modify germ cell production. of sexual activity, between September and December 1987. The work was the effect of a The aim of the present to analyse complete description of animals and methods of heating are mild scrotal in normal rams and in rams brief episode of heating given in a previous paper (Setchell et al, 1991). The pituitaries constant stimulation of the to dissociate the effects with testis, of ten rams (Hy) were removed surgically by\|he parapharyn- Received 23 March 1992. geal route, under halothane anaesthesia (Fluothan: Pitman Downloaded from Bioscientifica.com at 09/29/2021 12:48:31PM via free access Moore 3% in 02; 103mlmin_1). Each ram received a sup¬ ation, and on 10 cross-sections at stage 2 of the classification, plementation twice a day with ovine pituitary acetone extract during the elongation of the spermatid nuclei. These numbers in saline suspension at a dose of 120 mg for 19 days were not corrected for their size and consequently the total by intramuscular injection. One milligram of this powder is numbers of elongated spermatids per testis or of spermatozoa equivalent to 40 µg of NIH Si FSH, in a bioassay of FSH after spermiation were greater than those calculated for round according to Steelmann and Powley (1953). Five of the rams spermatids. At both of these stages (6—7 and 2, respectively), were submitted at the same time to hypophysectomy (HyH) the percentages of tubules with elongated spermatids or and local testicular hyperthermia, produced by immersing the testicular spermatozoa were calculated. shaved scrotum in a temperature-controlled water bath so that the rose 42 subcutaneous temperature to between and 43°C, Statistical analyses over 15 min and remained there for 45 min (four rams) or 30 min (one ram). There was no difference due to the time of All results were evaluated by two-way analysis of variance exposure in the effect on testis weight, and the results have with hypophysectomy-hormone treatment and heating as therefore been pooled. The other five hypophysectomized factors or by nonparametric U test (Mann and Whitney, 1947). rams were treated with pituitary extract but not heated (HyU). Three intact normal rams were submitted to scrotal hyper¬ thermia (IH; two for 45 min; one for 30 min, see above) and Results three were not heated (IU). All the Hy rams were given corti¬ sone (4 ml on day 1, 3 ml on day 2, 2 ml on day 3 and 1 ml on Testis weight, interstitial tissue volume and seminiferous tubule day 4 of a 25 mg ml-1 solution) and penicillin (Extancilline, parameters Specia 2.4 x IO6 units on day 1 and 1.2 106 units on days 2 and 3). On day 20 after heating or hypophysectomy, the Testis weight was significantly reduced by heat treatment, in animals were castrated under anaesthesia. both hypophysectomized HyH and intact IH rams, by 55 and 46%, respectively (Table 1). In addition, the testis was signifi¬ Histological and morphometric techniques cantly heavier in pituitary extract treated—hypophysectomized HyU than in normal intact IU rams ( + 33%). The relative All the analyses were performed on the right testis of each volume of seminiferous tubules in the testicular parenchyma ram, as no difference between the left and testes was significantly decreased by heating ( 8.4 and —9.4% significant right — was previously detected. Pieces of testicular parenchyma (1 or respectively; = 0.05) in HyH and IH rams. However, both 2 cm3) were fixed in Bouin Hollande solution for histological interstitial tissue and seminiferous tubules were affected by analysis. Analysis of intertubular and tubular tissues was per¬ heating. The total volume of interstitial tissue was reduced 44 and and formed as previously described (Hochereau-de Reviers et al, following heating by 32%, respectively, in HyH IH 1992). The relative volumes of intertubular tissue and of semini¬ rams. Total length of seminiferous tubule per testis was not ferous tubules were determined with a 25-point ocular integra¬ modified significantly by any treatment. The mean diameters of tor on 20 fields from each testis. The diameters of the seminifer¬ seminiferous tubules were significantly decreased by heat treat¬ ous tubules cut in cross-section were determined with a ment in IH ( 23%) or HyH ( 32%) rams and significantly graphic — — tablet analysis program (Apple Software) on 20 cross-sections increased in HyU ( +15%) compared with normal IU rams. As a per animal. The total length of the seminiferous tubules per result of variations in length and diameter of the seminiferous testis was calculated from the testis weight, the relative volume tubules, the surface areas of their basement membrane per of seminiferous tubules and the cross-sectional area of semini¬ Sertoli cell were significantly depleted by heating. The total ferous tubules (Attal and Courot, 1963) after correction for numbers of Sertoli cells per testis were not affected by any histological shrinkage. treatment, whereas their nuclear cross-sectional areas were The Sertoli cells, and significantly decreased by heat treatment ( 15% in HyH and A0, Ax spermatogonia, leptotene diplo¬ — tene and round were counted 9% in IH rams, respectively; < 0.05; Table 1). primary spermatocytes spermatids — in 10 cross-sections (10 µ thick) at stage 8 (spermatogonia and 1 and round spermatids), stage (leptotene), stage 3 (diplotene) of Germ cell the cycle of the seminiferous epithelium (Ortavant, 1959). For populations these cells, the corrected numbers of nuclei per cross-section, The total numbers of A0 spermatogonia per testis were not including those of the Sertoli cells that were assumed to be affected by any treatment (Table 2). Heating the testes induced spheres, were calculated by the formula of Abercrombie (1946). a significant fall, 20 days later, in the daily production of Aj The nuclear cross-sectional area of Sertoli cells was measured on spermatogonia, by 54 and 45% in HyH and IH rams, respect¬ 20 nuclei per animal at stage 8 (Ortavant, 1959). The total ively. Heating the testes induced a significant fall in the daily numbers of Sertoli and germ cells per testis were calculated production of leptotene primary spermatocytes, 20 days later, from the total length of seminiferous tubules per testis and the to the same extent as that of Aj spermatogonia ( 64 and — corrected numbers of germ cells per cross-section. The daily 30% in HyH and IH rams, respectively); gonadotrophin treat¬ — production of germ cells was calculated from their total ment of hypophysectomized rams significantly increased this numbers per testis divided by the duration of the seminiferous daily production in HyU rams compared with normal IU rams epithelium cycle, i.e. 10.4 days in the ram (Ortavant, 1959).