Effects of a single brief period of moderate heating of the testes on seminiferous 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 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 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 decreased by 95 and 90%, slightly more than the diplotene primary (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 . 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 , 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 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 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 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¬ 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). ( + 63%). However, the yields of spermatogonial divisions, Mean numbers of elongated spermatids were counted on 10 expressed as the ratio of leptotene primary spermatocytes upon cross-sections at stage 6—7 of classification, just before spermi- Aj spermatogonia, were similar in the four groups of rams.

Downloaded from Bioscientifica.com at 09/29/2021 12:48:31PM via free access Table 1. Testis weight and parameters of seminiferous tubules 20 days after a single brief episode of moderate heating of the testes in control or hypophysectomized pituitary extract-treated rams

Hypophysectomized (Hy) Intact (I) Significance of effect of Control HyU Heated HyH Control IU Heated IH Heat Hypo

Number of rams 5 5 3 Testis weight (g) 326 ± 21a 147 ± 22b 246 + 12c 132 ± 20" Interstitial tissue total volume (mm3) 47.7 ± 3.6a 26.7 ± 2.4b 37.3 ± 1.5a 25.6 ± 3.0h Seminiferous tubule: Relative volume (%) 77.1 ± 1.4a 70.7 ± 1.8b 76.3 ± 0.8a 69.2 ± 1.8" Mean diameter (µm) 266 + 7a 180 ± 14b 232 ± 8C 178 ± 14b Total length per testis (m) 2885 ± 277a 2484 ± 161a 2794 ± I22a 2366 ± 332a Sertoli cells: Total number per testis ( X 108) 27.9 ± 3.0 28.1 ± 2.1 27.2 + 2.6a 26.9 ± 3.0* Nuclear area (µ 2) 58.3 ± 2.7a 49.0 ± 3.4b 55.4 ± 1.2a 50.4 ± 0.5b Basement membrane area per 880 ± 53a 510 ± 53b 760 ± 58a 480 ± 44b Sertoli cell (µ 2)

Values are means + SEM. Hypo: Hypophysectomized and hormone treatment. Values that are not significantly different are followed by the same letter, whereas significantly different values are followed by different letters. '"P < 0.001, "P < 0.01, *P < 0.05.

Table 2. Germ cells parameters 20 days after a single brief episode of moderate heating of the testes in control or hypophysecto¬ mized pituitary extract-treated rams

Hypophysectomized (Hy) Intact (I) Significance of effect of Control HyU Heated HyH Control IU Heated IH Heat Hypo

Number of rams 3 3 A0 spermatogonia per testis (108) 3.7 ± 2.0a 2.5 + 1.2* 2.6 ± 1.1a 4.1 ± 1.8a Daily production per testis ( x 107) Aj spermatogonia 4.3 ± 0.5a 2.0 ± 0.3b 3.1 ± 0.6*b 1.7 ± 0.4b Leptotene primary spermatocytes 134 + 15a 48 + 25b 82 ± llab 58 ± 20b Diplotene primary spermatocytes 83 ± 6a 20 ± 8b 88 ± 12a 20 ± 9b Round spermatids 297 ± 31a 15 ± 15b 262 + 23a 27 ± 23b Total number per testis ( 108) Elongated spermatids 399 ± 42a 85 ± 59b 316 ± 20' 46 ± 10b Non-released spermatozoa 0.4 + o.y 32.6 ± 23b 0.7 ± 0.3a 10.3 ± 3.5b % of seminiferous tubule with 4 ± 3a 52 ± 19b 13 ± 9a 60 ± 12b spermatozoa (st2) Daily production of A1 spermatogonia 15.6 ± 1.1* 7.4 + 1.2b 11.3 + 1.9e 6.3 + 1.6b per Sertoli cell Yield of spermatogenesis Spermatogonial divisions Leptotene:Aj—meiotic prophase 31.8 ± 3.3' 23.3 10.9a 27.6 + 2.6a 33.1 ± 8.9a Diplotene:leptotene—meiotic divisions 0.7 ± 0.1* 1.5 0.9ab 1.1 ± 0.1" 0.4 + 0.1b Round spermatid:diplotene— 3.6 ± 0.3' 0.4 0.4b 3.0 ± 0.2* 1.0 ± 0.6b Elongated:round spermatid 1.4 ± 0.1* 1.4 + 1.1a 1.2 + 0.2* 2.6 + 1.1a

Values are means + SEM. Hypo: Hypophysectomized and hormone treatment. Values that are not significantly different are followed by the same letter, /hereas significantly different values are followed by different letters. *P < 0.05, "P < 0.01, '"P < 0.001.

Downloaded from Bioscientifica.com at 09/29/2021 12:48:31PM via free access Fig. 1. Seminiferous epithelium in adult rams (1 cm = 40 µ ). (a) Cross-section of a seminiferous tubule at stage 3 (st 3: classifi¬ cation of Ortavant, 1959) in a control ram; note the presence of elongating spermatids (eL), pachytene (P) and zygotene (Z) primary spermatocytes successively from the lumen to the basement membrane of the seminiferous tubule, (b) Cross-section of seminiferous tubules in hypophysectomized supplemented rams (Hy) at stages 6 (st 6) (upper) and 8 (st 8) (lower): note the presence of elongated (SZ) and round (R) spermatids, young pachytene (P) primary spermatocytes from the lumen to the basement membrane, (c) Cross- section of a seminiferous tubule at stage 1 in an intact heated ram: note the presence of non-released spermatozoa, of very few round spermatids, of pachytene (P) and leptotene (L) primary spermatocytes from the lumen to the basement membrane, (d) Cross- section of seminiferous tubules at stage 3 (upper) and 2 (lower) in an hypophysectomized heated ram (HyH); note the presence of some non-released spermatozoa (SZ) near the lumen, the absence of round or elongating spermatids, and pachytene (P) and leptotene (L: st 1) primary spermatocytes.

Daily production of diplotene primary spermatocytes Production of elongated spermatids was decreased but to a decreased significantly after heating the testes, more than the lesser degree than that of round spermatids; the percentage previous cell types, in both HyH and IH rams ( 76 and of stage 6-7 seminiferous tubules with elongated spermatids — 77%, respectively). dropped more drastically after heating in HyH ( 58%) than — — Daily production of round spermatids per testis, 20 days in IH rams ( —18%). However, in terms of total content of after heating, represented about 10% of the normal production elongated spermatids per testis, the decrease was similar in with no difference between HyH and IH rams ( 95 and IH ( 86%) and in HyH rams ( 79%). Moreover, normal — — — 90%, respectively); consequently the round spermatid rep¬ spermiation during stage 8 did not take place after heating and — resented the most depleted cell type. The yield of meiotic more than 20% of testicular spermatozoa were still present with divisions, visualized by the ratio of round spermatids to diplo¬ leptotene and diplotene primary spermatocytes in stage 2, in tene primary spermatocytes showed a significant fall, in both HyH and IH rams, whereas in IU or HyU rams, 13 and 4%, hypophysectomized pituitary extract-treated and intact rams. respectively of stage 2 seminiferous tubules presented some Downloaded from Bioscientifica.com at 09/29/2021 12:48:31PM via free access spermatozoa still loosely attached to the seminiferous epi¬ heating and remained low until about 40 days (Setchell et al, thelium. After heating, this was observed in 52 and 60% of 1971). Moreover, in the ejaculates, there was an increased pro¬ stage 2 seminiferous tubules in HyH and IH rams, respectively portion of dead or tailless spermatozoa between day 14 and day (Fig. 1). The non-released spermatozoa accounted for 38 and 50, indicating that all the germ cells from the AT spermatogonia 22% of the daily production of elongated spermatids in HyH onwards were affected by heating (Moule and Waites, 1963; and IH rams, respectively. Braden and Mattner, 1970). In mice, number of spermatozoa in epididymides fall from 2 to 5 weeks and assays for abnormal Correlations spermatozoa revealed damage from 1.5 to 6 weeks after heat exposure; this implies that a defect has occurred at the begin¬ The total volume of interstitial tissue was highly positively ning of spermatogonial multiplications (Cairnie and Leach, correlated with germ cell production, expressed per testis or per 1980). Aj spermatogonia and leptotene primary spermatocytes Sertoli cell: (r > 0.78; < 0.01) and to the basal membrane originate from cells that were Aj spermatogonia during the heat area per Sertoli cell (r = 0.89; < 0.01). treatment. By contrast, the A0 spermatogonia appeared not to be affected 20 days after heat treatment. They are reserve stem cells with a low labelling index after [3H]thymidine (Hochereau- Discussion de Reviers et al, 1976) and so presumably they are less sensitive to heating. The analysis of the ratios of leptotene primary The results of the experiment reported here are relevant to two spermatocytes to Aj spermatogonia revealed that heat shock different problems: the first is the long-term effect of a single killed the Aj spermatogonia rapidly but there is no progressive short heat treatment on seminiferous tubule function and the decay during successive spermatogonial divisions. The yield of second is the effect of high constant stimulation by pituitary meiotic divisions, estimated from the ratio of round spermatids hormones. to diplotene primary spermatocytes was very low: this could be Acute heat treatment of testes of rams has been said to affect, the consequence of the greater sensitivity of leptotene primary during the first 48 h, pachytene primary spermatocytes prefer¬ spermatocytes (giving rise to round spermatids 20 days later) as entially and round spermatids to a lesser extent; numbers of compared with type spermatogonia (giving rise to diplotene mitotic divisions of B: and B2 spermatogonia are increased, primary spermatocytes). probably resulting from an arrest of these divisions (Waites and The spermatozoa deriving from pachytene spermatocytes Ortavant, 1968). In the present experiment, twenty days after present at the time of heating showed a qualitative defect, as a single heat treatment, all the germ cells except the A0 sper¬ the normal release of testicular spermatozoa into the lumen matogonia were depleted, although elongated spermatids and did not take place; these unreleased spermatozoa are probably testicular spermatozoa were not completely absent. However phagocytosed by the Sertoli cells, later on. This implies that the heat treatment applied here was more intense, but of shorter either the microtubules, the membranes and the tubulobulbar duration (43°C for 30 to 45 min instead of 41.5°C for 3 to 4 h) processes or the trypsin-like enzyme concerned with this release than that used previously. Leptotene and diplotene spermato¬ (plasminogen activator) are abnormal after heat treatment. Such cytes, and round and elongated spermatids are derived from an abnormal release of spermatozoa was described by Swierstra cells that were reserve or cyclic A spermatogonia, spermato¬ et al (1964), in the rabbit after amphotericin treatment. A single gonia and pachytene primary spermatocytes, respectively, 20 episode of heating for 3 h at 40.5 °C induced abnormalities of days earlier. In the present experiment, the strongest quantitat¬ the internal structure of cytoplasmic droplets and of mitochon¬ ive effect seen after a delay of 20 days was not observed in dria (Voglmayr et al, 1971). Furthermore, even when some elongated spermatids, derived from pachytene spermatocytes, spermatozoa are produced, their ability to maintain embryonic but in round spermatids at stage 1, which were derived from development has been impaired after heating in rams (Mieusset leptotene primary spermatocytes at the time of heating, an et al, 1992). Increase in testicular enzymes (acid phosphatase observation not made previously, because only shorter times and leucine aminopeptidase) has been observed in rats after after heating treatment were studied. The leptotene stage scrotal heating (Blackshaw et al, 1973). Amino acid transport represents the period when DNA synthesis takes place in the (Hall et al, 1981), protein synthesis (Nakamura and Hall, 1980) primary spermatocytes (Ortavant, 1959) and it is the longest and dissociation of polysomes in ribosomes (Nakamura and S phase among the germ cells (Monesi, 1962; Hilscher, 1964; Hall, 1978) are heat-sensitive phenomena in rat spermatids. Hochereau-de Reviers, 1971; Huckins, 1971). This implies that All the observed effects of spermatogenesis could be associ¬ the proportion of germ cells in S phase per unit of time is highest ated with the presence of heat shock proteins in seminiferous for primary spermatocytes and decreases going backward to Aj epithelium, either in germ cells or in Sertoli cells (Maekawa et spermatogonia. DNA synthesis, in vitro, in human testicular al, 1989). In rats, Sertoli cells and germ cells from pachytene pieces, appears to be inhibited by an increase in temperature spermatocytes onwards contain heat shock proteins; no investi¬ (Nakamura et al, 1988). The spermatogonial multiplications gation was performed on younger germ cells, i.e. leptotene pri¬ appeared to be a thermosensitive phase of spermatogenesis in mary spermatocytes and spermatogonia. The HSP70.2-related the ram; this corroborates the effect of cryptorchidism in this proteins could be involved in the formation and disruption of species (Barenton et al, 1982). Furthermore, it was previously cytoskeleton proteins (Zakeri et al, 1988) and could play a role observed that after a single episode of heating, counts in in spermiation and explain the modification of spermatozoa the ejaculates of rams were low between 34 and 47 days after¬ formation and release. wards (Braden and Mattner, 1970); and the concentration of The Sertoli cells do not vary in number after heating; in spermatozoa in the fluid began to fall 20 days after cryptorchid rams, where the testicular temperature is increased Downloaded from Bioscientifica.com at 09/29/2021 12:48:31PM via free access to 39°C, arrest of spermatogenesis is observed without effect We are indebted to the animal hospital staff for the care of animals, on Sertoli cell numbers (Barenton et al, 1982). However, in the to M. Courot for providing the ovine pituitary acetone powder and to M. M. de Reviers for its FSH This work and the present experiment, the nuclear cross-sectional area of Sertoli assaying bioactivity. visit of B. P. Setchell in France were financed INRA and by a 'Bourse cells was in both groups of heat-treated rams, pre¬ by depressed de Séjours de Haut Niveau' from the French Government as the result of an effect of cell loss. A similar Scientifiques sumably germ to . P. Setchell. effect on Sertoli nuclear area was also observed in the sterile rat, in the absence of an increase in temperature (Viguier-Martinez et al, 1983) and is related to impairment of Sertoli function. In References rats after a single episode of heat, the Sertoli cell function, assessed by production of androgen-binding protein decreases Abercrombie M (1946) Estimation of nuclear population from microtome sections relatively rapidly after heating (Jegou et al, 1984). Anatomical Record 94 238-248 The amount of to Attal J and Courot M (1963) Développement testiculaire et établissement de gonadotrophin supplied hypophysecto¬ la chez le taureau Annales Animale, Biochimie, mized rams acetone extract was too and the spermatogenèse Biologie by pituitary high Biophysique 3 219-241 mean plasma concentration of FSH and LH were respectively Barenton B, Blanc MR, Caraty A, Hochereau-de Reviers MT, Perreau C and 3.5- and 7-fold higher after hypophysectomy than before Saumande J (1982) Effect of cryptorchidism in the ram: changes in the concen¬ in the same animals. Heat treatment did not trations of testosterone and estradiol and receptors for LH and FSH in the hypophysectomy and its Molecular and Cellular 28 13—25 modify these values (Setchell et al, 1991). The testis, histology Endocrinology high gonado¬ Blackshaw AW, Hamilton D and Massey PF (1973) Effect of scrotal concentrations account for increases in heating trophin presumably on testicular enzymes and spermatogenesis in the rat Australian Journal of testis weight, in seminiferous mean diameter and in daily Biological Sciences 26 1395-1407 production of leptotene primary spermatocytes compared with Braden AWH and Mattner PE (1970) The effects of scrotal heating in the ram on semen characteristics, and mortality Australian Journal normal rams the sexual season. 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Etude de son Contrôle de la treatment was too short to reveal increases in the older Expérimentale Gonadotrope·. Importance Lignée germ Sertolienne Thèse D. Sci., Paris cell or the fact that the number of cells population, by germ Hall PF, Mita M and Nakamura M (1981) Influence of temperature on amino acid (from zygotene primary spermatocytes onwards) engulfed in transport by round spermatids from rat testis Biology of Reproduction 25 each Sertoli cell is relatively limited and could not be 110-114 zur und der increased indefinitely. In the presence of concentrations Hilscher W (1964) Beiträge Orthologie Pathologie spermatogonio- high der Ratte zur Anatomie 130 69-132 of in but of numbers of genese Beiträge Pathologische gonadotrophins plasma, limiting Hochereau-de Reviers MT (1971) Etude cinétique des spermatogonies chez les could not be Sertoli cells, sperm production indefinitely mammifères: revue. In La Cinétique de Prolifération Cellulaire Vol. 18 INSERM increased, even in the absence of negative feedback. The Symposia Series Seminar, pp 189—216 Ed. M Tubiana. INSERM daily production of leptotene primary spermatocytes Hochereau-de Reviers MT, Ortavant R and Courot M (1976) Type A spermato¬ gonia in the ram. In Progress in Reproductive Biology: action Vol. 1 observed in our control pituitary Sperm hypophysectomized pp 13-19 Eds PO Hubinont and M L'Hermite. Karger, Basel extract-treated rams was identical that observed to in rams Hochereau-de Reviers MT, Monet-Kuntz C and Courot M (1987) Spermato¬ treated with 2-month light cycle regimens, which presented a genesis and Sertoli cell numbers and function in rams and bulls Journal of relatively low concentration of plasma gonadotrophins as¬ Reproduction and Fertility Supplement 34 101—114 Hochereau-de Reviers MT, Perreau Pisselet C and Pelletier Effect of a sociated with a low plasma testosterone concentration. 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