7-Alpha-hydroxytestosterone in seminiferous tubules of rat testis

Production of 4-androstene-7 alpha,17 beta-diol-3-one (7 alpha-hydroxytestosterone) from testosterone was measured in seminiferous tubule and interstitial fractions of rat testis. In adults, specific activity of the 7 alpha-hydroxylase was about 10 times higher in interstitial cells than in seminiferous tubules, but tubule production was a significant portion of the total. Seminiferous tubule 7 alpha-hydroxylase was not detectable in weanlings. Also, we confirmed that 7 alpha-hydroxytestosterone inhibits the activity of the 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase active on testosterone and suggest a role in maturational changes.     

Tamoxifen induced multinucleated cells （symplasts） and distortion of seminiferous tubules in rat testis

Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mg·kg-1·d-1, 400 mg·kg-1·d-1 or 800 mg·kg-1·d-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 μm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.     

Paracrine regulation of Leydig cells by the seminiferous tubules

Testes of adult control and unilateral cryptorchid rats were fixed by vascular perfusion. The cell profile area of peritubular Leydig cells surrounding tubules in different stages of spermatogenesis, and the cell profile area of perivascular Leydig cells were determined. The size of peritubular Leydig cells was dependent on which type of tubulus the cells were surrounding. Some peritubular Leydig cells, especially those surrounding stages VII–VIII (88.1 ± 7.1 μm², mean ± SD, n = 6 rats), were larger than perivascular Leydig cells (69.3 ± 5.9 μm²). The size of Leydig cells surrounding stages IX–XIV was similar to that of perivascular cells. In the abdominal testes no spermatogenic cycle was present and the sizes of peritubular and perivascular Leydig cells were equal (63.0 ± 5.1, vs 66.7 ± 7.3 μm², mean ± SD, n = 5 rats). It is suggested that the tubules and the spermatogenic cycle locally modulate Leydig cell activity and that Leydig cell malfunction in abdominal testes may be due to a decreased stimulatory influence from the damaged tubules.     

Development of stage-specific paracrine regulation of Leydig cells by the seminiferous tubules

The size of peritubular Leydig cells surrounding tubules in different stages of the spermatogenic cycle was determined in 43- and 47-day-old male rats. A stage-dependent variation in the size of peritubular Leydig cells was not present in 43-day-old rats, but by 47 days those Leydig cells closely adjacent to tubules at stages VII-VIII were larger than others. At 43 days of age spermatogenesis had developed up to step 18 spermatids in late stage VI tubules. At 47 days of age the first mature sperm had just been released from the seminiferous epithelium, and consequently the first wave of the spermatogenic cycle was completed. Tubules at stages VII-VIII therefore acquire the ability to influence surrounding Leydig cells when they contain step 19 spermatids. It remains to be shown whether this maturation step is due to inherent maturation of the Sertoli cells or if step 19 spermatids specifically modulate Sertoli cell function.     

大鼠曲细精管生殖细胞体外共培养和精子发生过程的观察

目的建立体外长期共培养体系和观察方法,为精子发生过程的研究提供细胞模型。方法采用大鼠曲细精管生殖细胞及支持细胞共培养的方法,对睾丸生精细胞作显微镜观察。结果共培养的支持细胞和生精细胞在体外存活超过6个月。在共培养期间,观察到精母细胞、圆形精子细胞和长形精子细胞。结论在不添加任何细胞因子和生长因子的情况下,大鼠曲细精管生殖细胞长期增生分化,不断产生精子细胞。这一结果暗示组织块和共生的支持细胞可为生殖细胞的增生和分化提供必需的细胞因子。该方法为体外研究精子发生过程提供了实验依据。     

Effects of lithium carbonate on rat seminiferous tubules: an ultrastructural study

Lithium salts are commonly used for treatment of bipolar disorder but prolonged treatment with therapeutic doses induces substantial toxic effects. In the present study we examined the effects of lithium carbonate on the ultrastructure of rat seminiferous tubules. Rats were exposed to lithium carbonate at doses of 35 mg/kg/day for 21 days. After lithium treatment, the tunica propria widened and folded together with convolutions of the basement membrane, myoid cells and lymphatic endothelium. In the seminiferous epithelium loss of germ cell attachment and appearance of expanded intercellular spaces between spermatogenic cells were observed. Early stages of spermatogenic cells showed nuclear protrusions or swellings because of an extensive enlargement of the outer nuclear membrane. Round spermatids exhibited abnormally shaped acrosomes and dilation of the subacrosomal space. Many abnormal, degenerated late spermatids with random orientation were seen towards the basal and adluminal compartments of the seminiferous epithelium. In addition, spermatids exhibited alteration in F-actin bundle ectoplasmic specialization and contained many mitochondria-associated granular bodies.     

Carcinoma-in-situ cells in cultured human seminiferous tubules

For the first time, early germ-cell derived tumour cells were studied in an in-vitro system of cultured seminiferous tubules. The intratubular tumour cells not only survived in culture for 7 days but were also able to multiply. Dividing tumour cells were identified in semi-thin sections and electron micrographs by morphological criteria. Additionally, mitotic activity was demonstrated by [3H]thymidine histo-autoradiography. There are numerous reports on cell lines established from solid non-seminomas, but up to now no references to seminoma cell lines or cultures of intratubular tumour cells are available. The culture of seminiferous tubules offers a tool in making carcinoma-in-situ cells accessible for experimental work.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

生精小管异位重构研究进展

哺乳动物睾丸的形成和精子发生是一个复杂的过程,包括细胞的迁移、增殖、分化和细胞间的相互作用。尽管近年来对此进行了大量的研究,但对许多方面仍然是未知。缺少一个合适的研究睾丸形成与精子发生的模型是阻碍研究取得进展的一个重要原因。在过去的几年中,相继发现2种对研究哺乳动物精子发生很有价值的模型:一是未成熟的睾丸组织异位移植于免疫缺陷鼠的皮下可以成活并产生有功能的精子;另一是未成熟睾丸组织消化成单细胞后异位移植于免疫缺陷鼠的皮下可以重新形成生精小管样结构并完成完整的生精过程。本文对这两种方法的研究进展、应用及前景进行了综述。     

Alterations in sperm characteristics of follicle-stimulating hormone (FSH)-immunized men are similar to those of FSH-deprived infertile male bonnet monkeys

The quality of sperm ejaculated by bonnet monkeys and normal, healthy proven fertile volunteer men, both actively immunized with ovine follicle-stimulating hormone (oFSH), was examined at different times of study for chromatin packaging and acrosomal glycoprotein concentration by flow cytometry. Susceptibility of sperm nuclear DNA to dithiothreitol (DTT)-induced decondensation, as measured by ethidium bromide binding, was markedly high compared with values at day 0 in men and monkeys during periods when FSH antibody titer was high. Sperm chromatin structure assay yields alphat values, which is another index of chromatin packaging. Higher alphat values, signifying poor packaging, occurred in both species following immunization with heterologous pituitary FSH. The binding of fluorosceinated pisum sativum agglutinin (PSA-FITC) to acrosome of sperm of monkeys and men was significantly low, compared with values at day 0 (control) during periods when cross-reactive FSH antibody titer was high and endogenous FSH was not detectable. Blockade of FSH function in monkeys by active immunization with a recombinant oFSH receptor protein corresponding to a naturally occurring messenger RNA (mRNA) also resulted in production of sperm with similar defects in chromatin packaging and reduced acrosomal glycoprotein concentration. Thus, it appears that in monkeys and men, lack of FSH signaling results in production of sperm that exhibit defective chromatin packaging and reduction in acrosomal glycoprotein content. These characteristics are similar to that exhibited by sperm of some class of infertile men. Interestingly, these alterations in sperm quality occur well ahead of decreased sperm counts in the ejaculate.     

De novo morphogenesis of seminiferous tubules from dissociated immature rat testicular cells in xenografts

Testicular development is initiated with the differentiation of Sertoli cells in the embryonic gonad. The aggregation of Sertoli cells is crucial for the generation of testicular cords and thus for the first sign of male gonadal development. To date, functional testicular tissue has not yet been generated in vitro. The objective of this study was to explore the de novo morphogenesis of testicular tissue from isolated postnatal rat testicular cells using a combination of in vitro culture and ectopic xenografting. Immature rat testicular cells were cultured in either a 2-dimensional (laminin-coated coverglass) or a 3-dimensional (extracellular matrix gel) culture system. Whereas testicular cells cultured on laminin showed a slow morphogenetic cascade resulting in cord formation after about 10 days of culture, cells cultured on extracellular matrix gel assembled to a network of cordlike structures within several hours after plating and formed spherical cell aggregates at day 3. Further progression of the morphogenetic cascade was not obtained in either the 2- or the 3-dimensional culture system. In contrast, structures resembling immature testicular tissue were obtained after xenografting of extracellular matrix gel-enclosed spherical testicular cell aggregates. The grafts were vascularized and contained elongated seminiferous tubules. Histologic analysis revealed the presence of a basement membrane, a histologically normal interstitium containing putative Leydig cells, the establishment of tubule lumen, and the integration of few putative spermatogonia into the seminiferous epithelium. We conclude that immature rat testicular cells carry the full potential to generate all somatic components of a testis in xenografts, thus opening fascinating pathways to study testicular organogenesis.     

Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo

In vivo microperifbsion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo . Somniferous and caput and cauda epididymal tubules were perihsed for 3 h. with [³⁵S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed.
Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules.
Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and path physiological effects on this important epithelial hnction in vim     

Differentiation of the rat seminiferous tubules between 13 and 19 days of age

The differentiation of rat seminiferous tubules have been studied in 13 to 19 days old rats. Testicular weight and tubular cross-sectional area were more than doubled during this period. The percentage of tubules with more than 10 primary spermatocytes increased from 4% to 90%, and the lanthanum excluding ability of the inter-Sertoli cell junctions (the blood-testis barrier) showed a similar increase but two days later. ABP production in vitro increased more than twentyfold from day 13 to day 19 of age. It is concluded that the differentiation of Sertoli cell function and the appearance of primary spermatocytes are temporally correlated which supports the assumption that the function of the Sertoli cells is important for initiation and maintainance of spermatogenesis.     

Multinucleated spermatocytes and spermatids in human seminiferous tubules

The cytology of multinucleated spermatocytes and spermatid giant cells in the seminiferous tubules of men with oligozoospermia and of men older than 65 years has been investigated electron microscopically. Two different processes which are responsible for the origin of multinucleated germ cells have been analysed: Defects of the intercellular bridges (IB): they move apart and thus allow the confluence of one clone's cells to symplasms. The confluence of membranes: within one clone the membranes of neighbouring germ cells are dissolved and thus intercellular bridges are found in the cytoplasm of the resulting giant cells. The spermatid giant cells reveal a new organization of the cell nuclei and the cell organelles. Yet they disintegrate in the lumen of the seminiferous tubules. The appearance of giant cells therefore is an expression of the germ cell degeneration.     

移植大鼠生精干细胞到裸鼠生精小管实验系统的建立

目的 建立移植大鼠生精干细胞到裸鼠生精小管的实验系统.方法 采用一次腹腔注射busulfan 30mg/kg以消除裸鼠生精小管内源性生精细胞,制备生精干细胞移植受体小鼠;采用laminin粘附的方法分离大鼠生精干细胞;应用生精小管直接注射和输出管注射的方法移植分离到的大鼠生精干细胞进入受体裸鼠的生精小管内.结果 busulfan腹腔注射4周后受体裸鼠生精小管中的精子发生明显得到抑制.大鼠生精干细胞移植2～3个月后在受体裸鼠的生精小管中发现了大鼠的长型精子细胞.结论 成功建立了移植大鼠生精干细胞到受体裸鼠生精小管的试验系统.     

Orchiectomy in young rats results in differential regulation of follicle-stimulating hormone and luteinizing hormone content

While it is generally accepted that GnRH stimulates release of pituitary gonadotropins, it is not clear what regulates synthesis. The orchiectomized immature rat, with sustained high plasma levels of LH and FSH, provides an opportunity to study how gonadotropin biosynthesis responds to loss of the gonad. We have measured plasma and pituitary LH and FSH in castrate and sham operated rats after orchiectomy at 15, 30, 45, and 60 days of age. Plasma FSH and LH concentrations by RIA were markedly elevated in castrates within one to three days after castration, and they remained elevated in all groups. By contrast, pituitary content measurements revealed differences between the two gonadotropins: while LH content in castrates consistently exceeded that in controls, FSH content in castrates was lower. Pituitary LH excess was evident by seven to ten days after castration. The pituitary FSH deficit in younger animals was similarly apparent by seven to ten days. In the older groups, however, FSH content decreased as early as three days, but returned toward normal by 21 days. In orchiectomized young rats, pituitary LH is elevated, but FSH content is depressed. This discrepancy is delayed, but more marked, in younger rats. In view of sustained high plasma levels after castration, our findings imply differential regulation of synthesis, processing, or storage of the two gonadotropins.     

Age dependent changes in FSH responsive adenylyl cyclase and FSH binding in rat testes

FSH responsive adenylyl cyclase (AC) activities were examined in both homogenates and membrane particles from 9–71 days old rat testes, and FSH binding was determined in membrane particles from the same animals. FSH responsive AC was measured in the presence of either GTP (0.04 mM) or the non-metabolizable GTP analogue, GMP-P(NH)P (0.04 mM). From days 9 and 11 there was a gradual decrease in both basal and FSH stimulated AC activities and a very similar decrease in FSH binding. The relative stimulation by FSH, however, was fairly constant (2–3 fold stimulation). The excellent correlation between specific FSH binding and FSH responsive AC activities during sexual maturation does not support the notion that uncoupling of the FSH receptors from the AC is a primary reason for the attenuated FSH response during pubertal development. The age dependent decrease in both basal and FSH stimulated AC activities during sexual maturation is a reflection of the relative increase in germ cells (containing no FSH receptors and FSH responsive AC). The LH/hCG responsive AC in membrane particles from the same ages showed an age dependent increase in both absolute activities and relative responses, probably indicating the increased number of Leydig cells in the developing testis.     

Transformation,migration and outcome of residual bodies in the seminiferous tubules of the rat testis

Experiments were performed to study the transformation, migration and outcome of residual bodies (RBs) in the seminiferous tubules of the rat testes. One part of the testes from adult Sprague–Dawley rats was used to generate paraffin sections to observe RBs and RB precursors through specific staining, and the other part of the testes was used to generate ultrathin sections to observe RBs under a transmission electron microscope. Deep blue particles of different sizes were observed in some seminiferous tubules through specific staining for RBs and RB precursors. These particles first appeared in the seminiferous tubules at stage I of the spermatogenic cycle, and after spermiation, the particles travelled rapidly towards the deeper region of the seminiferous epithelium and soon appeared close to the basement membrane of the seminiferous tubule. All of the particles in the tubules disappeared at stage IX. Using transmission electron microscopy, components of different electron densities were observed in the RBs on the surface of the seminiferous epithelium, all of which gradually formed in the cytoplasm of spermatozoon in later stages of spermiogenesis. After the spermatozoa were released, the RBs in the epithelium travelled quickly to the edge of the tube and were gradually transformed into lipid inclusions. These lipid inclusions ultimately became lipidlike particles. The lipidlike particles were discharged into the interstitial tissue. RBs initiate their own digestive process before their formation during spermiation in the rat testes. After spermiation, the RBs transform into lipid inclusions and finally into lipidlike particles. These lipidlike particles can be eliminated from the seminiferous tubules.