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The role of the proto-oncogene Kit expression during gonadal development, then in differentiated spermatogonia has been thoroughly established. The present study was designed to investigate the consequences of a partial defect in Kit gene expression on sperm fertilizing ability, using Kit haplodeficient mice (kitW-lacZ/+). Same inbred mice (kit+/+) were used as controls. Epididymal sperm characteristics and in vivo fertility were assessed, then in vitro-fertilization experiments were carried out for mice of both genotypes. Epididymal sperm count was drastically reduced, and sperm motility was also decreased in kitW-lacZ/+ compared with kit+/+ males. Both in vivo or in vitro fertility were greatly reduced in kitW-lacZ/+ compared with kit+/+ males. By contrast, the fertility of kitW-lacZ/+ females was apparently unaffected. Additionally, a higher number of spermatozoa with undetected acrosomal contents was revealed by fluorescein isothiocyanate-labelled Pisum sativum agglutinin acrosomal staining after epididymal sperm retrieval in kitW-lacZ/+ mice, whereas no difference was observed after induction of acrosomal reaction in mice of either genotype. Ultra-structural data confirmed the higher frequency of abnormal acrosome in spermatozoa of kitW-lacZ/+ mice. Thus, sperm production is impaired in Kit haplodeficient mice both on a quantitative and a qualitative basis. Finally, we show that one single copy of Kit gene is not sufficient to maintain genuine fertility in male mice.

Kit/stem cell factor (SCF ) has been reported to be involved in survival and proliferation of male differentiating spermatogonial cells. This kinetics study was designed to assess the role of Kit/SCF during spermatogenesis in mice, and the extent of male programmed germ cell death was measured between 8 and 150 days of age. For this purpose, 129/Sv inbred mice in which one Kit allele was inactivated by an nlslacZ sequence insertion (Kit(W-lacZ/+)) were compared with 129/Sv inbred mice with wild-type alleles at the Kit locus. Four different approaches were used: 1) morphometric study to assess spermatogenesis, 2) flow cytometry to study testicular cell ploidy, 3) in situ end labeling to detect apoptosis, and 4) follow-up of reporter gene expression. Spermatogenesis was lower in Kit(W-lacZ/+) heterozygous mice both at the onset of spermatogenesis and during adulthood. Indeed, greater apoptosis occurred at the onset of spermatogenesis. This was followed in the adult by a smaller seminiferous tubule diameter and a lower ratio between type B spermatogonia and type A stem spermatogonia in Kit(W-lacZ/+) mice compared with Kit(+/+) mice. These differences are probably related to the Kit haplodeficiency, which was the only difference between the two genotypes. Germ cell counts and testicular cell ploidy revealed delayed meiosis in Kit(W-lacZ/+) mice. Reporter gene expression confirmed expression of the Kit gene at the spermatogonial stage and also revealed Kit expression during the late pachytene/diplotene transition. These results suggest involvement of Kit/SCF at different stages of spermatogenesis.

The aim of the present analysis was to determine whether anti-Müllerian hormone concentrations in prepubertal plasma or adult rete testis fluid are related to the number or function of Sertoli cells in rams or to the presence of the FecB Booroola gene. Twenty rams from two Booroola crosses, differing in their testicular masses were analysed; in each cross, half of the animals were heterozygous carriers of the FecB gene. The data from rams, during prepuberty and at adulthood during the non-sexual season, were analysed by two-way ANOVA and residual correlations. In 4-week-old intact male lambs, the mean anti-Müllerian hormone plasma concentration was 15 ng ml-1, irrespective of cross, genotype or eCG stimulation; it was significantly negatively correlated with FSH (r = -0.51; P = 0.02; n = 19). In adults, anti-Müllerian hormone was not detectable in plasma and was 0.5 ng ml-1 in rete testis fluid, irrespective of cross or genotype. The total number of Sertoli cells per testis was not related to anti-Müllerian hormone concentration in lamb prepubertal plasma or in adult rete testis fluid. The concentration of anti-Müllerian hormone in adult rete testis fluid was significantly and negatively correlated with the daily production of leptotene primary spermatocytes per testis (r = -0.56; P = 0.02; n = 16). The mean oestrogen concentration in the adult testicular vein was 2 pg ml-1 and was correlated negatively with the rete testis fluid concentration of anti-Müllerian hormone (r = -0.60; P = 0.02; n = 15) and correlated positively with the daily production of leptotene primary spermatocytes per testis (r = 0.53; P < 0.05; n = 19). In conclusion, anti-Müllerian hormone secretion was not correlated with the total numbers of Sertoli cells per testis and cannot be used as a predictor of the number of Sertoli cells. Anti-Müllerian hormone secretions were not affected by the presence of FecB gene. However, anti-Müllerian hormone secretion could be considered to be inversely related to the daily production of primary spermatocytes by the testis.

The proliferation rate and differentiation state were investigated in porcine inner cell masses (ICMs) and epiblasts in vitro. ICMs isolated from early blastocysts (Day 7 of pregnancy) and epiblasts isolated from preelongated blastocysts (Day 11 of pregnancy) were cultured for up to 5 days in the presence of human leukemia inhibitory factor (hLIF) (1000 U/ml). The proliferation rate was evaluated by determination of the percentage of cells in S-phase. The differentiation state was determined by studying the expression of the stage-specific embryonic antigen-1 (SSEA-1), a marker for undifferentiated murine embryonic stem (ES) cells, and the expression of laminin and cytokeratins 8/18, markers of ES cell differentiation. The staining pattern showed that freshly collected Day 11 epiblasts appeared undifferentiated but rapidly lost this characteristic in vitro. A decrease in the proliferation rate was also observed during culture. This decrease was reduced in the presence of high concentrations of hLIF (optimal concentrations: 5000 U/ml). Conversely, treatment of Day 11 epiblast cells with retinoic acid, an agent known to induce differentiation in murine ES cells, caused a dramatic decrease in the proliferation rate in vitro. In contrast to Day 11 epiblasts, Day 7 ICMs expressed SSEA-1 in vitro and showed a higher proliferation rate (p < 0.01). However, their proliferation rate also decreased during culture and following trypsinization. These results indicate that the undifferentiated characteristics of Day 7 ICMs are more likely to be maintained in vitro than are those of Day 11 epiblasts, which are rapidly committed into early differentiation.

In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used: hypodactyl rat mutation (testis weight [TW]: 55% less than controls), in utero busulfan treatment (TW: 88% less than controls), and neonatal experimental cryptorchidism (TW: 72% less than controls). The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo. In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively. In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished. The addition of both control and damaged seminiferous tubule culture media (STM: media conditioned by 10 cm of seminiferous tubules) to 10(6) control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation. However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM. Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids. In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells.

In order to elucidate the respective effects of depletion of germ cells and of increase in testicular temperature, rats of the same Wistar strain were rendered experimentally bicryptorchid or sterilized by a busulfan injection in utero and compared to control animals. In both models, germ cells were depleted but numeric evolution and functions of somatic cells differed. The aim of that work was to compare the numeric evolutions of testicular somatic and germ cells to their respective functions in each model before puberty and in adult rats of the same strain. Serum concentrations of FSH, LH and testosterone were compared at 20, 40 and 110 days of age. Histological analyses of Sertoli and germ cells in the seminiferous tubules and of Leydig cells in the intertubular tissue were performed before puberty and at adulthood. Testosterone serum concentrations were depleted in both models starting at 40 days of age and more in busulfan-treated rats. Both FSH and LH levels were increased from 20 days onwards in experimental rats. Additional cryptorchidism in busulfan-treated rats depressed the serum testosterone concentration. At 17 days of age, the cryptorchidism do not modify somatic or germ cell populations while busulfan treatment has induced a decrease of both these populations. Conversely, the cross sectional area of the somatic testicular cells was not affected whatever the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Testicular development of sheep fetuses was studied between day 42 of gestation and birth. Testis mass and the total number of testicular cells increased curvilinearly with fetal age and a positive linear relationship was established between the logarithmic values of age and testis mass, sex cord total length, total number of Sertoli cells, germ cells and Leydig cells per testis. The mean number of gonocytes per unit length of sex cord, the Sertoli cell nuclear cross-sectional area and the Leydig cell cross-sectional area decreased linearly with age between day 42 of gestation and birth. Hypophysectomy and hemicastration were performed to study the regulation of testicular cell divisions during fetal life and to determine whether they were under pituitary control and whether a feedback mechanism was present. Hypophysectomy at day 100 or 110 of gestation nonsignificantly decreased (0.05 < P < 0.01) the testis mass, total length of sex cords and total number of Sertoli cells and significantly decreased (P < 0.05) the cross-sectional area of Leydig cells and nuclei of Sertoli cells. Sex cord diameter and total number of gonocytes were unaltered. Hemicastration at day 110 of gestation significantly increased (P < 0.05) the total number of Leydig cells per testis without changing any other testicular parameter. In male sheep fetuses, the proliferation of testicular somatic and germ cells occurs throughout testicular fetal growth at a higher rate before day 100 of gestation than later, but without any differentiation. Mitotic divisions of Sertoli cells are more numerous before birth than afterwards. Before birth, the proliferation of gonocytes is not under pituitary control.

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 degrees C for 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 modifications 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 multiplications 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of Trypanosoma congolense on testis was studied in 53 trypano-resistant "Baoulé" bulls by quantitative histology and morphometry. The daily spermatozoa production per testis of control groups (n = 45) was 382 +/- 334 x 10(6) (m +/- sd) and the epididymis contained 0.6 +/- 1 x 10(9) spermatozoa in the caput, 0.3 +/- 0.3 x 10(9) in the corpus and 1.2 +/- 1.8 x 10(9) in the cauda. The infected bulls (n = 8) showed no significant difference (P > 0.05) when compared to the control despite their average low value. The morphometric analysis during infection revealed a significant (P < 0.05) decrease (32%) of total Leydig cell volume per testis, 4.4 +/- 0.9 cm3 for the control (n = 5) and 3.0 +/- 0.8 cm3 for infected bulls (n = 8). The number of round spermatids per Sertoli cell and the daily round spermatid production (DRSP) per testis were also significantly reduced in infected bulls when compared to controls (P < 0.05), 5.2 +/- 0.7 and 2.8 +/- 2 for round spermatid per Sertoli cell and 6.1 +/- 2.0 and 3.1 +/- 1.9 x 10(8) for DRSP. These observations indicate that Trypanosoma congolense infection alters the interstitial tissue and meotic divisions of germinal cells leading to low daily round spermatid production per gram of testis in "Baoulé" bulls.

The addition of seminiferous tubule (ST) culture medium (STM) prepared from testes of either busulfan-treated (Bus) or cryptorchid (Cryp) or genetically sterile (hd) rats, to Percoll purified Leydig cells leads to a further increase of LH-stimulated testosterone (T) output (26, 43 and 14%, respectively). Taking into account that the Sertoli cell number per cm of ST is 2.6, 1.8 and 1.4-fold greater in Bus, Cryp and hd rats than in controls, the above STM effects on T output, expressed per 10(6) Sertoli cells are in fact lower (63, 44 and 43%, respectively) that those of control STM. Similar results have been obtained for the STM transferrin levels which are decreased, 74, 67 and 45%, respectively in Bus, Cryp and hd animals. So, it is likely that the Sertoli cell secretion of both the paracrine factor involved on Leydig cell T production and the transferrin is influenced mainly by spermatids and to a lesser extent by spermatocytes of mature rat testis.

This experiment was conducted in Ile-de-France adult rams to examine the target point of a 2-month light cycle regimen on seminiferous tubule functions, on intertubular compartment and on Leydig cell parameters. Eight rams were subjected to a 2-month light cycle regimen and were compared to sexually active or inactive rams. In light-treated rams, testis weight was maintained equal or was higher than that of sexually active rams. Both tubular and intertubular tissues were found significantly higher in light-treated than in sexually active rams. The mean ratio of basement membrane area of the seminiferous tubules per Sertoli cells and the daily productions of A1 spermatogonia and of leptotene primary spermatocytes were significantly increased in light-treated rams as compared with sexually active or inactive rams. Meanwhile, the dairy productions of diplotene primary spermatocytes, of round spermatids, of spermatozoa and of the rete testis fluid were not significantly increased in light-treated as compared with sexually active rams but significantly greater than those of sexually inactive rams. Total volume, total numbers, and individual volumes of Leydig cells were at least equal or higher in light-treated than in sexually active rams.

The morphology and in-vivo function of the Leydig cells were studied in rams when spermatogenesis had been disrupted by a single exposure of the testes 20 days earlier to a temperature of about 42 degrees C for 45 min. To avoid complications due to changed negative feedback from the testes to the pituitary with consequent changes in the degree of gonadotrophic stimulation, ten of the animals (five heated and five unheated) were surgically hypophysectomized when the testes were heated and then treated twice daily with pituitary extract. Six intact rams (three heated and three unheated) were also studied. The heat-affected testes were about half the size of the unheated testes, and blood plasma flow was closely related to testis weight. There were no differences in the testosterone concentrations in spermatic venous blood, testicular lymph or rete testis fluid, or in oestradiol in spermatic venous plasma from heated or unheated testes. Consequently, testosterone secretion by the heat-affected testes was markedly reduced, and the concentrations in jugular blood were also lower in the heat-affected rams than in controls. The volume of the interstitial tissue was less in absolute terms in the heat-affected rams, but it made up a greater fraction of the testes. The absolute volume of the blood plus lymph vessels, and their fraction of the interstitial tissue were lower in the heat-affected testes, although there was no effect on their volume as a fraction of the whole testis. The heat-affected testes of the hormone-treated rams had fewer Leydig cells, but each cell was larger; no equivalent difference was found in the intact rams. However, the dose of pituitary extract chosen was somewhat excessive, as there were higher than normal concentrations of FSH, LH and testosterone in jugular blood plasma, of testosterone and oestradiol in testicular venous blood plasma and of testosterone in rete testis fluid in the hormone-treated hypophysectomized rams. The testes of the unheated hypophysectomized rams increased in size by about 20% during treatment with pituitary extract, although testicular blood plasma flow was lower per unit weight of testis. The absolute volume of each Leydig cell and the total volume in absolute terms and as a fraction of the interstitial tissue was greater in the hormone-treated than in the untreated rams, but not the volume as a fraction of the whole testis. The total number of Leydig cells was higher in the hormone-treated unheated rams than in all the other rams taken together.(ABSTRACT TRUNCATED AT 400 WORDS)

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular cyclic AMP and testosterone productions by purified mature rat Leydig cells were stimulated by oLH (25 micrograms/l) 18- and 12-fold, respectively, after a 5-h incubation period. The replacement of the incubation medium by charcoal-treated testicular venous plasma (40%, v/v) from adult rams in the breeding season induced an inhibition of cyclic AMP and testosterone productions (82 and 66%, respectively, of oLH-stimulated values). Testicular arterial plasma is less effective than testicular venous plasma, even when it originates from non-breeding season rams; in that case, testicular venous and arterial plasma strongly inhibit testosterone productions (84 and 67%, respectively of oLH-stimulated values), which probably indicates that the inhibitory activity is higher in the non-breeding season. The addition of peripheral plasma leads to a testosterone production equal to 35 and 65% of the oLH-stimulated values, respectively, for ram blood collected in non-breeding and breeding seasons. The same concentration of ovine testicular lymph or rete testis fluid is without significant effect on cyclic AMP production; however, testosterone is slightly decreased by lymph but enhanced by rete testis fluid. Increasing amounts of venous or arterial testicular blood induce a dose-related decrease of the specific binding of labelled hCG in both rat and ram testicular membranes. This inhibiting factor is present in peripheral and testicular blood of either control or hypophysectomized or castrated rams, is a protein in nature, heat-sensitive, and has an apparent molecular weight higher than 10,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

In mature rat Leydig cells, the testosterone output (24 ng/10(6) Leydig cells/4hrs.) is increased 10 fold by LH; the addition of serum from either control or castrated or hypophysectomized rams inhibits (60%) the LH-stimulated testosterone production. Similarly, the incubation of immature rat Leydig cells with sera from hypophysectomized patients leads to a diminution (70 and 30% respectively) of both basal (0.98 ng) and LH stimulated (3.44 ng) testosterone biosynthesis. These data suggest the existence of an LH inhibitor (or inhibitors) in blood from ram and human; in addition, this substance is not only of testicular origin and is not an LH-related molecule.

Endocrinological and testis parameters of adult 18-month-old Ile de France (IF) and Romanov (Ro) rams were compared during sexual season. Testis weights, total volumes of intertubular tissue, and of blood and lymph vessels, total seminiferous tubule length, rete testis flow rate and daily production of germ cells were significantly higher in IF than in Ro rams. These variations originated from differences in Sertoli cell numbers, which were established before puberty. When daily productions of germ cells, of ABP or of RTF were expressed per Sertoli cell, they were higher in Ro than in IF rams. Quality of spermatids, as measured by their cellular size prior to elongation, was lower in Ro than in IF. The number of FSH-binding sites per Sertoli did not differ between the two breeds but FSH plasma levels were higher in Ro than in IF rams. Total numbers of Leydig cells per testis, their individual size or their LH-binding capacity did not differ significantly between the two breeds. However, the ratio of mean testosterone upon mean LH plasma levels were greater in Ro than in IF rams while both breeds had identical LH mean plasma levels.

Dwarf mice show delayed testicular growth and their adult testis weights are half the normal value. The aims of the present work were firstly, to compare the developmental profiles of plasma gonadotropins and of testicular cell multiplication and differentiation in dwarf vs normal mice and secondly, to determine the effect of hMG supplementation on dwarf mice. In the dwarf mice no pubertal rise in plasma FSH was observed, and the adult values remained very low when compared with those of normal mice; plasma LH decreased after 40 days of age and remained equal to half the normal values. In adults, testicular testosterone content was greatly increased in dwarf mice compared with normal mice, whereas plasma testosterone and accessory gland weights were reduced. At 24 days of age, the total numbers per testis of Leydig and Sertoli cells were reduced in dwarf vs normal mice, whereas in adult mice their differentiation, but not their total numbers, was reduced. This resulted in lower daily production of leptotene primary spermatocytes and of round spermatids in dwarf than in normal mice. hMG supplementation promoted Leydig and Sertoli cell multiplication, but did not produce full differentiation, resulting in increased daily production of round spermatids. In conclusion, in adult dwarf mice a deficiency in plasma gonadotropins prevents full differentiation of Leydig and Sertoli cells without affecting the number of these cells.

In order to investigate the effects of induced hypoprolactinaemia on gonadotrophin levels and testicular function in the ram, CB 154 was administered either in summer under natural photoperiod or in winter combined with a light regime stimulating prolactin release. Under natural photoperiod in summer, plasma FSH levels increased from 2.5 to 4 ng/ml in controls and from 2.5 to 8 ng/ml in CB 154-treated rams. Control and treated groups differed significantly (P less than 0.05) from the second week of treatment onwards. In the animals photostimulated in winter, treatment with CB 154 did not change plasma FSH levels; no effect of CB 154 on plasma LH levels was detected in either experiment. Treatment with CB 154 in summer led to an increase in the volumes of peritubular blood vessels and interstitial tissue. The number of Leydig cells per testis was significantly (P less than 0.05) increased in CB 154-treated rams. However, at the time of measurement, there was no change in the structure of the seminiferous tubules or in the production of germ cells. In winter in photostimulated animals, no effect of treatment with CB 154 was detected either on the structure of intestitial and tubular compartments or on spermatogenesis. The change in prolactin levels affected neither the number of LH receptors, expressed per Leydig cell, nor the number of FSH receptors, expressed per Sertoli cell, in the experiments. It is concluded that induced hypoprolactinaemia can modify the release of FSH in the ram and change the structure of the intertubular tissue but that it does not affect the number of LH and FSH receptors or impair spermatogenesis.

Changes in testicular androgen receptor numbers were studied in lambs from 25 to 100 days of age. During this period, cytoplasmic receptors increased from 5 to 80 pmol/testis and nuclear receptors from 1 to 12 pmol/testis, while the total volume of Leydig cells increased 7-fold. The total number of Sertoli cells doubled between 25 and 40 days of age. From 40 days onward their number remained constant while their cellular and nuclear sizes increased by a factor of 3 and 1.5 respectively. Cytoplasmic receptor concentration was positively correlated with the number of Sertoli cells per section of seminiferous tubule, and negatively correlated with the number of germinal cells per cross section. One explanation for these results could be that Sertoli cells are the main androgen target cells in lamb seminiferous tubules.

Continuous low dose gamma irradiation induces a progressive degeneration of germ cells with a concomittant increase in blood FSH; however, the Sertoli cell function is not too much altered since serum ABP level is normal and it is likely that the decrease of epididymal ABP content is the consequence of a reduction in seminiferous tubule fluid excretion. Obviously, spermatids seems to be involved in the regulation of Sertoli cell ABP synthesis.

A histological study of the testes of stags shot in autumn (sexual season) and spring (quiescent period) indicated that the 3-fold increase in testicular size observed in the autumn was accompanied by increases in nearly all features studied (volumes of intertubular tissue, Leydig cells, blood vessels and peritubular cells; diameter and length of seminiferous tubules; the number of A1 spermatogonia and products of spermatogonial divisions, meiosis and spermiogenesis). There were, however, fewer A0 spermatogonia in the testes in autumn.

Plasma gonadotrophin and testosterone levels during the prepuberal period have been compared in spring and autumn-born Romanov lambs; the somatic and germ cell populations of these lambs have also been compared at birth and at adulthood. At 1 and 3 months of age, the number of LH pulses per hour and mean plasma LH levels were significantly higher in spring than in autumn lambs. In adults, no such differences were observed. Similarly, at 1 and 3 months of age, the number of testosterone pulses per hour was higher in spring than in autumn animals. In the prepuberal period, the ratio of the levels of mean plasma testosterone to LH was higher in autumn than in spring lambs. The highest ratio was observed in adults but there was no variation according the season of birth. At 1 month of age, mean plasma FSH levels were higher in autumn than in spring lambs; this difference did not persist later on. Despite these endocrinological differences, testis weight, total Sertoli and Leydig cell numbers per testis, total number of gonocytes per testis at birth and daily production of round spermatids per testis in adult rams were similar in spring and autumn-born animals.

The reversibility of damage caused by cryptorchidism to the seminiferous tubules of the lamb was investigated at various ages. Lambs were made bilaterally cryptorchid either at birth or at 2 months of age. Then orchidopexy was performed at either 2 or 4 months of age. In permanently cryptorchid lambs, spermatogenesis stopped completely, and Sertoli cell function, as measured by FSH receptors, androgen receptors and ABP, was much reduced (-96%, -86% and -81%, respectively). Orchidopexy allowed the cryptorchid seminiferous epithelium to grow again, but the more differentiated the germ cells, the less they were capable of restoration. Even in 0- to 2- and 0- to 4-month-old temporarily cryptorchid lambs that had recovered normal Sertoli cell function, 16 to 49% of the tubules still were empty. It was concluded that cryptorchidism irreversibly damages the seminiferous tubules at a level other than the hormone receptors.

We have found evidence in the male lambs, Booroola x Mérinos d'Arles and Booroola x -Romanov, during the first three months of life, for the expression of the "F" prolificacy gene. More FSH at the peripheral plasma levels is observed in males carrying one copy of the "F" gene as compared to non carriers of the same crosses in the same environment. However the difference is of too small amplitude to be utilised to discriminate male lambs carrying the "F" gene.

Production of testosterone and oestradiol-17 beta by Leydig cells from adult rats was stimulated by LH or dibutyryl cyclic AMP (10 and 2.5-fold respectively). The addition of spent medium from normal, hemicastrated or gamma-irradiated rat seminiferous tubule cultures, as well as from Sertoli cell cultures, to purified Leydig cells further enhanced both basal (44 and 53% for testosterone and oestradiol-17 beta respectively) and LH-stimulated (56 and 18%) steroid output. Simultaneously, a decrease (20-30%) in intracellular cyclic AMP levels was observed. This stimulating factor (or factors) secreted by the Sertoli cells is different from LHRH, is of proteinic nature and has a molecular weight ranging between 10,000 and 50,000; its synthesis is not controlled by FSH nor by testosterone. This factor(s) involved in rat Leydig cell steroidogenesis, at a step beyond the adenylate cyclase, does not require protein synthesis for testosterone formation whereas it does for oestradiol-17 beta production. It should be noted that a germ cell-Sertoli cell interaction modulates the synthesis of this factor(s).

A rete testis cannulation technique was used to compare the secretion of rete testis fluid with the production of spermatozoa and histological testicular parameters in Ile-de-France rams. Thirteen testes were cannulated and rete testis fluid variables were compared to histological variables of the same testis. The rate of flow of rete testis fluid was significantly (P less than or equal to 0.05%) correlated with testicular size, the area of the walls of the seminiferous tubules and the volume of the Leydig cells. These two latter factors accounted for 66% of the variation in the flow of rete testis fluid.

Production of spermatozoa and secretion of rete testis fluid (RTF) in rams was assessed by a rete testis cannulating technique. Four breeds (Ile-de-France, Romanov, Préalpes du Sud and cross-breed Romanov) were studied throughout the year. Inhibitory effects of the cannulation process on spermatogenesis were observed for some animals. Between-breed differences were found in sperm concentration and flow rate of the RTF. The seasonal variations in the daily sperm production of the testis were more pronounced for Ile-de-France rams than for the other breeds. There was a seasonal variation in the flow rate of RTF in Ile-de-France rams, the minimum flow being in February (winter) and the maximum in August-September (autumn).

Active immunization of Ile-de-France rams against estradiol (E2) resulted in the production of E2-neutralizing antibodies and an elevation in the plasma concentrations of FSH, LH, and testosterone. The presence of E2 antibodies did not affect the testosterone metabolic clearance rate, indicating that the immunization-mediated 10-fold increase in plasma testosterone was the result of a 10-fold increase in testicular testosterone production. Testis weights, as well as nuclear and cytoplasmic volumes of individual peritubular and perivascular Leydig cells, were greater in E2-immunized rams than in albumin-immunized controls. Leydig cell numbers were not affected by treatment. The E2 antibodies were capable not only of neutralizing the inhibitory effects of endogenous E2 on gonadotropin levels in intact rams, but were able to block the effects of exogenously administered E2 on their FSH and LH secretory response to castration. It is concluded that circulating E2 in the ram is involved in pituitary-testicular endocrine homeostasis and that E2 immunoneutralization can be employed to enhance testosterone secretion in this species.

Adult male rats received one injection (J0) of busulfan (10 mg/kg BW), which is specifically lethal for type A spermatogonia (Jackson and al., 1962). They were subsequently injected daily (J1 - J10) either with a proteinaceous extract of adult ram testis (10 mg of proteins/day) via the subcutaneous route, or with ram testicular fluid (1, 2 mg of protein/day), or with bovine serum albumin (1 mg/day) in order to analyse the resultant modification of the repopulation of seminiferous epithelium by the reserve stem cells (Clermont and Mauger, 1974). At the end of the treatments (J11), the number of type A spermatogonia per tubular cross section was significantly decreased in animals receiving testicular extract of fluid. The number of primary spermatocytes, from preleptotene up to zygotene stages were only diminished by testicular fluid supplementation. Ten days later (J20) the populations of primary spermatocytes, from preleptotene to pachytene stages derived from cells which were type A spermatogonia at the time of injections, were depleted. Type A spermatogonia were only decreased by testicular fluid treatment. Testicular fluid or extracts did not modify body weight nor the weights of other organs. A protein-like factor originating from the testis and present in the testicular fluid can thus modify the population of type A spermatogonia. Its action, however, is not specific for germ cells as Sertoli cells are affected by the administration of testicular extract in prepuberal rats (Hochereau-De-Reviers and Courot, 1978).

Female Wistar rats were treated with busulfan or with solvent on Day 20 of pregnancy. Thirty male offspring of each group were killed at 38 days of age. In busulfan-treated rats, compared to controls, hypothalamic LH-RH content was decreased by 52%, whereas pituitary LH and FSH concentrations were increased by 60 and 43% respectively. Plasma LH and FSH were increased by 112 and 275% respectively. Prolactin concentrations were not changed, but plasma testosterone concentration was decreased by 48%. The total number of Leydig cells per testis was decreased by 52%, and LH binding sites per testis were decreased by 70%. The total number of Sertoli cells was decreased by 44%, while FSH binding sites per testis were decreased by 62%. Spermatogenesis was practically absent after prenatal exposure to busulfan. These data demonstrate that on Day 20 of pregnancy all the dividing cells in the fetal testes were depleted by an antimitotic treatment. The stimulation of the hypothalamo-pituitary axis could have been partly induced by the decrease in testosterone production, and by the aplasia of germ cells involving modifications of the remaining Sertoli and Leydig cells.

The weight and histology of the testis and plasma LH levels were analysed after hemicastration of adult Ile-de-France rams in spring or in autumn. After hemicastration, the remaining testes were significantly heavier than those of entire animals measured at the same time of year. At 4 or 6 months after hemicastration performed in spring, the remaining testes were hypertrophied by nearly 40% as compared to the testes of entire sexually active animals, assessed in autumn. The variations of intertubular tissue volume, total seminiferous tubule length, stem cell stocks, daily production of round spermatids, and cellular volume of primary spermatocytes paralleled the variations in testis weight. The annual decrease of the area of the Sertoli cell nuclei and of the yield of meiosis and beginning of spermiogenesis during the non-breeding season was prevented by hemicastration performed in autumn. Plasma LH levels were consistently elevated till autumn after hemicastration performed in spring. A positive and significant correlation was observed between LH levels and yields of spermatogonial divisions.

Cryptorchidism was induced in 5 pre-pubertal lambs and 7 adult rams, 5 months after surgery, testicular weight and membrane protein content were 4-fold lower than in the control. The total number of Leydig cells per testis was markedly decreased but their size was not changed. In contrast, the total number of Sertoli cells per testis was not affected but their nuclear size was smaller. Induced cryptorchidism had no effect on the length of seminiferous tubules; blood vessel volume was reduced; and the production of germ cells was completely disrupted. The number of LH receptors estimated per Leydig cell was not changed in pre-pubertal lambs but decreased 4-fold in adult rams. The number of FSH receptors calculated per Sertoli cell was reduced by 95% in both pre-pubertal and adult animals. No effect on the binding affinities of LH (Ka = 1 X 10(10) M-1) and FSH (Ka = 4.5 X 10(9) M-1) to their testicular receptors was observed. Although testicular concentrations of testosterone and estradiol-17 beta were increased, the total content of testosterone within the testis was increased only in pre-pubertal lambs. The estimated ratio of testosterone per Leydig cell was higher in cryptorchid animals than in controls, suggesting that, despite their reduction in number and the decrease of LH receptors, the Leydig cells of cryptorchid rams have an enhanced steroidogenic capacity. This study also confirms the important dysfunction of the Sertoli cells in cryptorchid rams.

Two types of stem spermatogonia- type A0 and A1 spermatogonia- have been observed in ram and bull testes. Type A0 spermatogonia are present in the impuberal testis. Their total number/testis does not vary greatly during life of bull. Type A0 spermatogonia give rise to type A1 spermatogonia which appear at the onset of spermatogenesis; their total number/testis increases during and after puberty. Total numbers of type A0 and A1 spermatogonia/testis are not correlated in the ram while total numbers of type A1 spermatogonia and Sertoli cells are correlated. Hemicastration performed in impuberal calves induces an increase in numbers of Sertoli cell/type A1 spermatogonia/testis. In adult ram, the total number of A0 and A1 spermatogonia decreases during the non sexual season and increase again in the sexual season. After hypophysectomy performed in adult rams, the number of A0 spermatogonia/testis is not modified while that of A1 spermatogonia decreases. In ram and bull, 6 spermatogonial generations were detected before the germ cells undergo meiosis. In the adult ram and bull, the type A1 spermatogonia originate mostly after divisions of A2 spermatogonia. Few of them arise from A0 spermatogonia. The yield of primary spermatocytes from spermatogonial divisions is very low, one third and one half of the expected number in the bull and ram respectively. In ram, thus yield positively correlated to LH plasma levels (0.5; P = 0.05). In hypophysectomized ram, this yield is nearly equal to zero.

LH, FSH and testosterone variations were compared during the prepubertal period in Romanov and Ile-de-France ram lambs born in the spring. Mean plasma LH levels increased significantly between the 1st and 8th week of age in the Romanov and between the 1st and 12th week of age in the Ile-de-France. At 1 month of age, the number of LH and testosterone pulses per hour was higher in Romanov than in Ile-de-France lambs. The mean plasma testosterone levels, higher in the Romanov, increased from 1 week of age onwards. Mean plasma FSH levels increased from the first neonatal week till the 8th week in the Romanov and till the 12th week in the Ile-de-France. The levels of FSH did not differ significantly between the two breeds. The higher and earlier secretion of LH and production of testosterone in the Romanov might be a cause of its better reproductive performance.

Six adult rams were hypophysectomized and immediately injected with 0,5 g (group A) or 2 g (group B) of testosterone per day for 2 weeks. Three normal rams (group C) received the solvent only. The testosterone concentrations in the testis (ng/g) were the same in groups A (23 +/- 7) and C (22 +/- 4) and 3 times higher in group B (72 +/- 18). There was an uptake of testosterone by the testis of the two supplemented groups. In the Rete Testis Fluid, the testosterone concentration was normal with 0,5 g/day (29 +/- 0.6 ng/ml). The testicular weight was maintained by the two treatments. However, spermatogenesis was abnormal in both supplemented groups; meiosis and spermiogenesis occurred normally, but the efficiency of spermatogonial divisions, as shown by the number of leptotene and zygotene primary spermatocytes, was greatly reduced (85%). The results indicate that testosterone alone in the Ram is unable to support complete spermatogenesis, which is contrary to that found in the Rat.

Adult male Wistar rats were treated with flutamide from 90 to 105 days of age. In a first experiment, testis and accessory sex organs were weighed. In the same animals, hypothalamic LRH content, pituitary gonadotrophin concentrations, plasma LH, FSH, prolactin and testosterone levels, and testicular gonadotrophin receptors were evaluated. In a second experiment, fertility was tested at the end of the treatment, and histology of the testis was performed. All the results were compared to those obtained in control animals of the same age. Accessory glands of genital tract were significantly lower in flutamide-treated animals (P less than 0.01). Hypothalamic LRH, pituitary and plasma FSH, and prolactin concentrations were unchanged, while pituitary and plasma LH level and especially plasma testosterone concentration were increased (P less than 0.001). Flutamide therefore exerted a strong inhibition on testosterone-dependent organs, and blocked the negative feedback of testosterone on the hypothalamo-pituitary axis, increasing the LH levels. Testis weight, intertubular tissue volume, total number and total volume of Leydig cells/testis, as well as total length and diameter of seminiferous tubules were unchanged in flutamide treated rats. However number of LH receptors/Leydig cell, nuclear area of Sertoli cells, number of FSH receptors/Sertoli cell, number of leptotene spermatocytes and of round spermatids per cross section, and yield of spermatogonial divisions were decreased after treatment. Flutamide treatment also decreased fertility by 48% (P less than 0.05). This lowered fertility is likely the result of impaired spermatogenesis and/or a dysfunction of accessory sex organs.

The two main types of cellular associations (type I, 2 generations of spermatocytes + 1 of spermatids; type II, 1 of spermatocytes and 2 of spermatids) occupy, respectively, more than half and about a third of the seminiferous epithelium cycle in rams and bulls. However, the duration of the cycle of the seminiferous epithelium and that of spermatogenesis differ between the species. A1 spermatogonia and Sertoli cell total numbers are highly correlated in adult rams and bulls. Mitosis in Sertoli cells occurs mostly in utero but may still occur for a short period after birth. Between birth and puberty there is about a 5-fold increase in the number of Sertoli cells. After that there are no seasonal- or age-related increases in the number of adult Sertoli cells. Some factors (season of birth; nutrition; genetics; hormones) affect mitosis of Sertoli cells in prepubertal animals. Sertoli cells differentiate after cessation of mitosis. Their differentiation is affected by cryptorchidism, nutrition, genetics and hormones. Their adult function is only poorly known. ABP and rete testis fluid secretions and nuclear Sertoli volume fluctuate under the influence of the same factors, but they are not always linked together. This reinforces the need for more knowledge of Sertoli cell secretions and function.

Male rats treated prenatally with busulfan in order to render them aspermatogenic were treated between 23 and 38 days of age either with testosterone, or flutamide. In such aspermatogenic rats, testosterone supplementation stimulated the growth of accessory sex organs, markedly decreased the secretion of gonadotrophins (by 61% for LH and 83% for FSH), decreased testicular weight (-60%) as well as all parameters relating to testicular histology. Flutamide treatment decreased the weight of accessory sex organs, stimulated the secretion of both LH (+200%) and FSH (+63%) by inhibition of the negative feedback of testosterone; endogenous testosterone secretion was increased by 363%. Testicular weight was increased by 44% and the total volume and the cytoplasmic and nuclear area of Leydig cells were increased by 254 and 28%, respectively, but their number per testis was unchanged. The number of Sertoli cells per testis was increased by 49%, but their nuclear area was not modified. In aspermatogenic prepubertal rats, the large increase in plasma FSH demonstrated that FSH release was partly under the control of inhibin, secreted by Sertoli cells when germinal cells were present. Nevertheless, FSH levels after testosterone (inhibition) or flutamide treatment (stimulation) clearly demonstrated that, in the growing rat, FSH secretion is also partly dependent on the negative feedback of testosterone. In the absence of germ cells, FSH was able to re-initiate Sertoli cells mitoses, but not their functional activity. LH secretion was solely controlled by the negative feedback of androgens and, even in the absence of germinal cells, Leydig cells were able to respond to LH stimulation by an increase in testosterone secretion.

Sertoli cells are involved in the control of spermatogenesis. In adult animals, the Sertoli cell stocks, fixed before puberty, are highly correlated with those of the germ cells. Some factors influencing Sertoli cell stock formation have been analyzed: 1) Early hemicastration of impuberal animals showed that the proliferative activities of the Sertoli and the germ cells before puberty, rather than their initial numbers at birth, were the limiting factors of their final number in adult testis. 2) During testicular growth, LH and FSH levels were correlated to Sertoli cell stocks and to germ cell production. 3) The season of birth influenced the Sertoli cell stock formation in the ram. Daylength modified testicular development in the cockerel and the sheep. 4) Between-strain differences were more marked for the testis than for body growth in the cockerel. In the ram, Sertoli cell stocks were similar between half-brothers but significantly different between sires.