Heterogeneity in end-terminal sugars of rabbit and rat androgen binding protein (ABP)

The interaction between rabbit and rat androgen binding protein (ABP) and rabbit serum testosterone binding globulin (TeBG) with concanavalin A (Con A) was studied using affinity chromatography on Con A-Sepharose 4 B columns. When partly purified rat ABP, equilibrated with [³H]5α-dihydro-testosterone [³H]DHT was applied to Con A-Sepharose columns, approximately 50% of the ABP was retained by the column, whereas the remaining was eluted with the break-through protein fraction. A similar picture was found using partly purified rabbit ABP, or crude rabbit rete testis fluid. These studies indicate that both rat and rabbit ABP are glycoproteins, showing heterogeneity in their end-terminal sugars. When partly purified rabbit TeBG was examined by Con A-Sepharose affinity chromatography, the TeBG was completely retained by the column. The different elution patterns between rabbit ABP and rabbit TeBG indicate that these proteins, although showing identical physico-chemical and immunological properties (Weddington et al. 1975a, b). possess differences in their carbohydrate content.     

Age-dependent Sertoli cell responsiveness to germ cells in vitro

This study investigated the effect of germ cells (greater than 80% mid- and late-pachytene spermatocytes) on the secretion of androgen binding protein (ABP) and transferrin by monolayer cultures of Sertoli cells isolated from rats aged 10, 18 or 26 days. There was an age-dependent increase in secretion of ABP and transferrin. Treatment of the Sertoli cell monolayers with hypotonic buffer to remove residual germ cells reduced this increase significantly. On the other hand, addition of germ cells to hypotonic-treated Sertoli cell monolayers increased both basal and FSH + testosterone-stimulated ABP and transferrin secretion at all three ages, although Sertoli cells from 10-day-old animals showed the greatest response. Moreover, addition of germ cells reduced responsiveness to FSH + testosterone in Sertoli cell monolayers obtained from rats aged 18 or 26 days. In monolayers obtained from 10-day-old rats, the opposite effect was noted in the case of ABP secretion. The stimulatory effect of germ cells on ABP and transferrin secretion was proportional to their number, and was reversed 48 h after the germ cells added previously were removed by hypotonic treatment. Whereas the reversal was complete with cultures of Sertoli cells isolated from 18- and 26-day-old rats, approximately 40% of the stimulatory effect remained after removal of germ cells from cultures from the 10-day-old age group. Adhesion of germ cells to Sertoli cell monolayers was also found to be age-dependent, with the largest proportion of added germ cells adhering to Sertoli cells isolated at 18 and 26 days of age. It is concluded that germ cells can significantly and differentially modulate the basal and hormone-stimulated secretory activity of Sertoli cells in vitro and that Sertoli cell responsiveness to germ cells (pachytene spermatocytes) is age-dependent and seems to appear early during the maturation process, before these germ cells appear in the testis.     

Studies on the scrotal testis in unilateral experimental cryptorchidism in rat and guinea pig

The effects of experimental unilateral cryptorchidism on the scrotal testis regarding weight, morphology and secretion of tubular fluid and the sertoli-cell specific androgen binding protein (ABP) were studied. In the intact guinea pig testis and epididymis an androgen binding component similar to rat ABP was found. In juvenile and adult rats cryptorchid for 17 and 21 days, respectively, and in guinea pigs cryptorchid for 11 weeks, the scrotal testis seemed unaffected regarding all parameters studied. With reference to previous findings of lowered fertility in unilateral cryptorchidism in man the possible mechanisms by which unilateral cryptorchidism may influence the scrotal testis are discussed.     

Cytokines produced by microwave-radiated Sertoli cells interfere with spermatogenesis in rat testis

Microwave radiation resulted in degeneration, apoptosis or necrosis in germ cells at different stages. The molecular mechanisms by which microwaves induce spermatogenesis disorder have not been completely understood. Sertoli cells play crucial roles in mammalian spermatogenesis. Cytokines produced by Sertoli cells play pleiotropic roles in different conditions. At physiologically low concentration, TNFα, IL-1β and IL-6 behave as survival factors; while under pathological condition, these cytokines can induce apoptosis in testis. The effects of cytokines produced by microwave-radiated Sertoli cells on spermatogenesis are poorly understood. The purpose of this study was to determine the effect of cytokines produced by microwave-radiated Sertoli cells on the germ cells. We focused the effect of TNFα, IL-1β and IL-6 on the germ cells. The results showed that TNFα, IL-1β and IL-6 were increased in Sertoli cells after exposure to microwave radiation. These up-regulated cytokines can induce apoptosis and lipid peroxidation in the membrane of germ cells. In addition, germ cell apoptosis was associated with the up-regulation of Bax/Bcl-2 and caspase-3. These results suggest that cytokines produced by microwave-radiated Sertoli cells may disrupt spermatogenesis. Our data provided novel insight into the injury mechanism of germ cells induced by microwave radiation.     

Involvement of apoptosis in the control of Sertoli and pre-meiotic germ cell numbers in the developing rabbit testis

The numbers of Sertoli and pre-meiotic germ cells in the developing rabbit testis were investigated as an initial step in determining the physiological meaning of the control of cell number in the testis by apoptosis. Sections were stained immunohistochemically for the detection of apoptotic cells and counterstained with haematoxylin. The number resulting from subtraction of the number of apoptotic cells from the total cell number was defined as the viable cell number. The number of viable premeiotic germ cells in the adluminal compartment of seminiferous tubules decreased during the pre-natal period, although neither apoptotic nor necrotic figures were detected. After birth, the numbers of total and apoptotic Sertoli and pre-meiotic germ cells increased, maintaining a stable ratio of their viable cell populations until the induction of meiosis. During induction of meiosis, the increase in the number of viable Sertoli cells was significantly accelerated because of the rapid decrease in the number of apoptotic Sertoli cells. Just after spermatids were formed the number of viable spermatogonia increased, reflecting an active supply of differentiated sper matogonia entering meiosis. In conclusion, apoptosis of Sertoli and pre-meiotic germ cells plays an important role in the acquisition of a suitable ratio of both cell types, and in providing intratubular environments for further progression of spermatogenesis, by controlling numbers of both cell types during the post-natal period.     

Maturation of the juvenile rat testis after surgical treatment of cryptorchidism

Rats were made bilaterally cryptorchid at 17 days of age and bilateral orchidopexy performed at 34 days of age. The epididymal content of androgen binding protein (ABP), the weight and morphology of the testis, the cross-sectional area of seminiferous tubules and the testicular concentration of testosterone were then studied at 34, 42, 59 and 120 days of age. Cryptorchidism was followed rapidly by progressive inhibition of spermatogenesis and testicular growth as well as by decreased Sertoli cell secretion of ABP. Orchidopexy resulted in a gradual restoration of spermatogenesis, and all impaired parameters seemed to improve at the same, fairly slow rate. Restoration was not complete, but by 120 days of age the morphological appearance of the testis was compatible with recovery of normal fertility.     

Carcinoma-in-situ of the testis and invasive growth of different types of germ cell tumours. A revised germ cell theory

The hypothesis is proposed that seminomas and non-seminomas are histogenetically closely related and both types of germ cell tumours may originate from a common precursor cell: namely the germ cell showing the carcinoma-in-situ pattern. However, it is suggested that the spermatocytic seminoma is an exception as it may originate from spermatocytes.     

Dose and time related responses of the irradiated prepubertal rat testis

Dose-related effects of radiation upon the seminiferous epithelium were examined in the prepubertal rat. Marked damage to the spermatogenic cell populations was produced by doses of 5 Gy and above. As a result serum levels of FSH were increased, maximum changes being observed at 2 weeks, but levels remaining high even at 36 weeks after irradiation. Serum levels of androgen binding protein (ABP) were inversely related to serum FSH. Above a threshold dose of 5 Gy, serum ABP levels were reduced to between 30-50% of control values, possibly indicating damage to the Sertoli cells. Progression in the degree of testicular failure was indicated at 36 weeks after a dose of 15 Gy by further increases in serum gonadotrophins when compared to earlier times and by the absence of spermatogenesis.     

Determination of Sertoli cell numbers in the developing rat testis by stereological methods

Stereological studies were performed to determine the number of Sertoli cells present during the postnatal development of the rat testes. Sprague-Dawley rats aged from 1 to 70 days were used in two experiments, and in each were fixed by vascular perfusion and embedded in Epon-Araldite, subsequent to which 1 micron sections stained with Toluidine blue were prepared. In the first experiment, rats aged from 1 to 20 days were used in groups of three, and number estimates were made using a direct counting method. In the second, which used groups of four rats aged from 20 to 70 days, a point sampled intercept was used to estimate nuclear volume and thence number. The results of the experiments indicate that the newborn rat testis contains 1.3 +/- 0.2 x 10(6) Sertoli cells and that this number increases to 38.4 +/- 2.7 x 10(6) at day 15. No further increase in Sertoli cell number occurred thereafter up to day 70 of age.     

Selective uptake by the Sertoli cells of cytoplasm from normal spermatogonia in the rat testis

Electron microscopical examination of germ cells during their development from early type A spermatogonia to late pachytene spermatocytes showed that small, spherical pseudopodia emerged from type B spermatogonia and, to a lesser degree, from intermediate spermatogonia and early spermatocytes. Serial sections showed that the pseudopodia pinched off from the type B spermatogonia and were engulfed by the adjacent Sertoli cells. Groups of dense bodies were found in the Sertoli cells adjacent to the engulfed islands of germ cell cytoplasm. At a few instances islands of germ cell cytoplasm were seen to fuse with dense bodies in the Sertoli cells. The fate of the cytoplasmic islands is unknown, but phagocytosis by the Sertoli cells may be suggested. The findings indicate a new type of interaction between Sertoli cells and certain classes of spermatogonia.     

Changes in intratesticular testosterone, cytoplasmic androgen receptors and ABP content of the ram testis after a single endogenous pulse of LH

The effects of an endogenous LH pulse on the testicular concentration of testosterone, cytoplasmic androgen receptor content and androgen binding protein (ABP) content was studied in rams. Blood was collected from 36 rams for at least 14 h and their testes then removed. Animals were then grouped according to the interval from their last LH pulse and the time of slaughter. The concentration of testosterone in the testis was highest during the first h following the LH pulse and returned to its baseline within 5 h. There was a 60% reduction in the testicular content of cytoplasmic androgen receptors at 3 h after the LH pulse, followed by slow replenishment. The testicular content of ABP was highest at 4 h after the LH pulse. It is in the testis concluded that the pulsatility of LH and testosterone induces a pulsatile translocation of cytoplasmic androgen receptors.     

Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules

In order to test the hypothesis that Leydig cell function in the adult rat testis is influenced by the surrounding tubules, Leydig cell morphology was compared in different types of interstitial areas. Triangular interstitial areas surrounded by 3 cross-sectioned tubules in nearly the same stage of spermatogenesis were chosen for quantitative light microscopy. It was found that the volume density of Leydig cells in such areas was about 30%, except when the surrounding tubules were in stages IX-X or XI-XII, when it was only about 20%. This variation in total Leydig cell mass seemed to be due to a variation in Leydig cell size and not in Leydig cell number. The largest Leydig cell profile area, 118 pL 6 μm² (mean pL SE n = 6 rats), was observed when the surrounding tubules were in stages VII-VIII, i.e. just prior to sperm release. The smallest Leydig cells were seen when the surrounding tubules were in stages IX-X and XI-XII (68 pL 3 and 66 pL 4 μm²). The present results indicate that there may be a Leydig cell cycle in the adult rat testis, which is regulated by the adjacent tubules.     

In-vitro influence of germ cells on Sertoli cel l-secreted proteins: a two-dimensional gel electrophoresis analysis

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was used to analyse [³⁵S]-methionine-labelled proteins secreted in vitro by Sertoli cells when cultured in the presence or absence of enriched preparations of pachytene spermatocytes (SPC), early spermatids (SPT) or residual bodies/cytoplasts from elongated spermatids (RB/CES). The presence of germ cells modified the pattern of Sertoli cell secreted proteins in co-culture. Out of 21 Sertoli cell secreted polypeptide families visualized by 2D PAGE, one (referred to as number 12) was stimulated, whereas the secretion of polypeptides 1 and 3 was inhibited by all of the germ cell populations tested. Early spermatids and RB/CES both enhanced the secretion of protein number 10 and inhibited the production of protein 11. The RB/CES fraction specifically inhibited secretion of polypeptide 13. Of particular note was the finding that co-culture with early spermatids or RB/CES induced the secretion of a novel polypeptide, termed GIP (germ cell-induced protein), with an apparent molecular weight of 72 kDa and an isoelectric point of 5.9. Under the present experimental conditions, media conditioned by the different germ cell fractions inhibited the secretion of polypeptide 2 but enhanced the secretion of polypeptides 10 and 18; of note also was the finding that media conditioned by early spermatids or RB/CES induced the appearance of GIP. This study confirms and extends the concept that germ cells influence Sertoli cell function and that the effects observed differ according to the stage of development of the germ cells. However, the sensitivity of the 2D gel electrophoresis technique, and to some extent its reproducibility, limit its use for studying the paracrine control of Sertoli cells in culture.     

B2 bradykinin receptor mediates the stimulatory effect of bradykinin on rat germ cell proliferation in vitro

The contribution of B1 and B2 bradykinin receptors to germ cell proliferation was studied by using in vitro organ cultures of testicular fragments of 3.5-day-old rats in the presence of 3H thymidine. Different combinations of agonists and antagonists of B1 and B2 receptors exerted differential mitogenic effect on pro-spermatogonial cells. Application of bradykinin (B2 receptor agonist) alone induced a threefold increase in germ cell labelling index compared with the control, whereas des-Arg9-bradykinin (B1 receptor agonist) caused weak stimulation (24%) on spermatogonial mitotic activity. The bradykinin-induced germ cell proliferation was significantly affected by B2 receptor antagonist (HOE-140) but not by B1 receptor antagonist. When B1 or B2 receptor antagonists were applied with des-Arg9-bradykinin, the germ cell labelling indices were nonsignificantly different compared with those of B1 receptor agonist only. The present findings suggest that B2 receptor is involved in mediating the stimulatory effect of bradykinin on germ cell proliferation and therefore bradykinin might be an important local testicular factor in the regulation of spermatogonial division and germ cell number.     

Recovery of testicular functions after surgical treatment of experimental cryptorchidism in the rat

When rats were made unilaterally cryptorchid at 17 days of age (before spontaneous descensus), the further maturation of the testis was prevented. At 34 days of age, the abdominal testis was smaller than the scrotal testis and showed less secretion of the Sertoli cell specific androgen binding protein (ABP). In 120–130 days old rats that were made bilaterally cryptorchid at 17 days of age, testicular weight, histology, secretion of fluid and ABP were restored and testosterone secretion and fertility were normal if orchidopexy was performed at 33 days of age. If the orchidopexy was delayed until 59 days of age, the recovery of testicular function and morphology was only partial. The results show that in the rat, the testicular damage caused by cryptorchidism is reversible, if the abdominal testis is surgically descended during early sexual maturation.     

大鼠支持细胞雄激素结合蛋白mRNA水平的生后变化

本文对大鼠睾丸支持细胞雄激素结合蛋白ｍＲＮＡ水平的生后变化进行了动态观察。研究表明，大鼠生后２天睾丸支持细胞即具有表达雄激素结合蛋白的能力。以后随着日龄的增长，ＡＢＰｍＲＮＡ水平轻度减少。进入青春期以后，ＡＢＰｍＲＮＡ水平开始增加，于生后２５天达到高峰。随之经过一短暂低水平后，支持细胞ＡＢＰｍＲＮＡ水平于出生４０天之后再次缓慢升高。