mRNAs encoding a von Ebner's-like protein and the Huntington disease protein are induced in rat male germ cells by Sertoli cells

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.     

Appearance of the oocyte activation ability of mouse round spermatids cultured in Vitro

This study was undertaken to determine the expression time of fertilization and oocytes activation abilities of spermatids in the mouse. When elongating or elongated spermatids isolated from fresh testes of adult males (B6D2F1) were injected into mature mouse oocytes, both spermatids could activate the mature oocytes and occur fertilization. On the one hand, the round spermatids could not activate mature oocytes, when microinjected into oocytes. In some experiments, recovered round spermatids were cultured under co-culture systems using Sertoli cells as a feeder cell. Under the co-culture system, developed elongating spermatids could stimulate and fertilized mature oocytes. These results indicate that the start of oocyte activation appearance is between the stage of round spermatid and elongating spermatids and the activation ability increases with the advance of spermiogensis. On the other hand, round spermatids isolated from males of ICR strain mouse already have the oocyte activation ability and the fertilizing ability. The result obtained suggests that the expression time of the oocyte activating ability is difficult between the mouse strain.     

Role of follitropin receptor signaling in nuclear protein transitions and chromatin condensation during spermatogenesis

Follitropin receptor (FSHR) in testicular Sertoli cells mediates signaling by pituitary follitropin (FSH) promoting intercellular communication with germ cells for normal spermatogenesis. Using receptor knockout mice we examined changes in sperm nucleoproteins and chromatin architecture. The expressions of transition proteins 1/2 (TP1/2) and protamine-2 (PRM-2) were greatly diminished at 21 days, but returned to normal at 35 days and 3 months after birth. However, protein components in chromatin were quite different. Western blots detected a reduction in PRM1/2 and prolonged retention of mono-ubiquitinated histone 2A (uH2A) in the epididymal sperm from adult mutants. Two forms of mono- and poly-uH2A were present in sonication-resistant testicular spermatids in normal mice, whereas only an elevated mono-uH2A was detectable in mutants. Decrease in PRM1/2 and retention of mono-uH2A was coincident with reduction in TP1/2 in premature spermatids. Thus lack of FSHR signaling impairs expression of TP1/2 and PRM-2 at an early stage of post-natal development causing delayed spermatogenesis. In the adult, absence of FSHR signaling prolongs retention of mono-uH2A, leading to impair transition of basic nucleoproteins and chromatin remodeling during mouse spermatogenesis.     

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

Culture of in vitro fertilized bovine embryos with bovine oviductal epithelial cells, Buffalo rat liver (BRL) cells, or BRL-cell-conditioned medium

Co-culture with various cell types can enhance development of bovine embryos, especially through the transition from maternal to embryonic mRNA utilization, a stage of growth refractory to most in vitro methods. Bovine oviductal epithelial (BOE) cells have been particularly successful for culturing embryos through the refractory stage; however, Buffalo rat liver (BRL) cells are a readily available, long-lived, easy-to-care-for alternative. This study compared the embryotrophic activity of BOE to BRL cells with particular emphasis on the transition stage of growth. A total of 7158 immature bovine oocytes, matured and fertilized in vitro, were divided into 4 different culture treatments: Treatment 1: BRL conditioned medium for 72 h then BRL co-culture; Treatment 2: BRL co-culture; Treatment 3: BOE co-culture for 72 h in 5% oxygen then BRL co-culture; and Treatment 4: BOE co-culture for 72 h in 5% oxygen followed by BOE co-culture in air. Those same treatments were used to evaluate embryotrophic differences of early (4 to 5) versus late (14 to 15) passage BRL cells maintained in M-199 medium with 10% serum. Two bulls were also evaluated to determine if there exists a bull-by-culture system interaction. Treatment 3 resulted in the best development after 9 d; 9.1% of selected immature oocytes developed to expanded blastocyst. Early passage BRL cells were significantly more embryotrophic than later passage cells; this was most pronounced for Treatment 2. There was a treatment-by-bull interaction, which should be considered when comparing results among similar studies.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.     

Developmental potential of mouse follicular epithelial cells and cumulus cells after nuclear transfer.

The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 microm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.     

Immunohistochemical localization of testicular sulphydryloxidase during sexual maturation of the Djungarian hamster (Phodopus sungorus)

Immunohistochemical localization of sulphydryloxidase was examined in the testis of the Djungarian hamster from Day 0 to Day 31 of post-natal development. The sulphydryloxidase antibody labelled prespermatogonia and the first population of spermatogonia type A within the seminiferous epithelium. Additionally, Sertoli cells exhibited immunoreactivity from Day 2 to Day 11 after birth. From Day 11 onwards, sulphydryloxidase immunoreactivity was found in germ cells after the initiation spermatogenesis from pachytene primary spermatocytes, showing the highest intensity in mid-pachytene spermatocytes. The pattern of sulphydryloxidase expression during spermatogenesis was identical to that found in adult animals. It is concluded that sulphydryloxidase immunoreactivity not only serves as a marker for early stages of spermatogenesis, especially pachytene spermatocytes, confirming earlier reports, but also for spermatogonial precursors.     

Ultrastructure of gametogenesis in the ovotestis of an estuarine pulmonate slug,Onchidium tigrinum (Stoliczka, 1869)

The pulmonate slug Onchidium tigrinum (Stoliczka, 1869) is an estuarine protandrous gastropod. Transmission electron microscopy of both the gonadal and somatic cell populations of the ovotestis of the slug is documented. The acini of smaller slugs are comprised of developing spermatogenic cells and three to four small ill-developed oocytes. Details of the microscopic structures of Sertoli cells, interacinar cells and acinar boundary are described in-depth, revealing their secretory function. Sertoli cells are more numerous in the ovotestes of smaller slugs than in those of larger slugs. Tunnelling nanotubes of 200–400?nm in diameter are described for the first time in the Sertoli cells of molluscan ovotestis. These nanotubes may help to supply various cellular materials into distantly developing spermatogenic cells. The acini of larger slugs possess 2–3 mature oocytes along with a few spermatogenic cells. Sertoli cells, interacinar cells and spermatogonial cells are fewer in number in the acini of the ovotestis of larger individuals establishing the predominance of oogenesis in this phase of life. The number of oocytes per acinus is analysed in relation to the habitat of the pulmonates.     

Protandric hermaphrodite peculiarities in Amphiprion frenatus Brevoort (Teleostei, Pomacentridae)

Electron microscopy of the male phase of the ovotestis of Amphiprion frenatus , a protandric hermaphrodite, showed no connective tissue between male and female areas and, as the basal lamina was lacking both along the seminiferous tubules and round the previtellogenic oocytes, the male and female germ cells were only separated by their respective surrounding somatic cells (Sertoli and follicle cells). Besides previtellogenic oocytes, oocytes in meiotic prophase and very small (young) previtellogenic oocytes, were detected in the ovarian part, as spermatogenesis proceeded, revealing oogenetic activity. Degeneration of some previtellogenic oocytes and their follicle cells was discernible.     

Sertoli细胞饲养层对小鼠精原干细胞增殖的影响

摘要 目的 以小鼠睾丸支持细胞(Sertoli)为饲养层,小鼠胚胎成纤维细胞(STO) 饲养层做对照,研究它对小鼠精原干细胞增殖的影响。方法 用无血清StemPro-34 SFM培养基培养2～5日龄小鼠精原干细胞,分别用相差显微镜观察,免疫组化法研究Sertoli饲养层对精原干细胞生物学行为的影响。结果 发现精原干细胞在Sertoli及STO两种饲养层上的一周内的生物学行为非常相似,但培养1周后,Sertoli细胞作饲养层的培养体系中保留的精原干细胞要比对照组明显增多,约有30%的精原干细胞能存活下来并能维持存活到60d以上。结论 Sertoli细胞作饲养层明显促进精原干细胞的更新增殖。     

Intratesticular androgen levels, androgen receptor localization, and androgen receptor expression in adult rat Sertoli cells

In the rat, quantitatively normal spermatogenesis is maintained only when intratesticular testosterone (ITT) levels greatly exceed the peripheral T concentration. When ITT concentrations fall below a threshold, germ cells are lost at specific stages of the seminiferous cycle. Germ cells can be restored by high doses of T that binds to androgen receptors (AR) in Sertoli cells. However, the relationships between germ cell dynamics, AR-mediated molecular events, and ITT concentrations are not established. ITT levels may regulate germ cell life and death through an effect on AR localization and AR mRNA or protein levels within Sertoli cells at specific stages of the cycle. We determined AR localization and mRNA and protein expression in adult rat Sertoli cells in relation to reduced and then restored ITT concentrations in vivo. ITT levels were reduced by implanting rats with T- and estradiol (E)-filled capsules for 7-28 days and subsequently restored with large T-filled capsules. AR is normally localized within Sertoli cell nuclei at stages VII-VIII of the seminiferous epithelium. After T/E treatment, AR immunostaining in Sertoli cell nuclei became nondetectable by 14-28 days but was restored 6 h following T restoration. The loss of Sertoli cell nuclear AR localization correlated with increasing numbers of apoptotic germ cells. AR mRNA levels in isolated Sertoli cells did not change through 14 days of T/E treatment, increased significantly by Day 28, and remained elevated 24 h after T restoration. AR mRNA levels in microdissected tubules at stages II-IV, VI-VIII, and IX-XII did not decrease through 14 days of T/E treatment. In contrast, AR protein levels were reduced in seminiferous tubules by Day 14 and in testes at Day 28 post-T/E treatment but were restored within 24 h by T repletion. Therefore, the reduction of ITT concentration results in a time-dependent redistribution of AR and reduced AR protein but not AR mRNA levels in Sertoli cells. Repletion of T restored AR protein and it relocated to Sertoli cell nuclei. By an unknown mechanism, T regulates AR localization within Sertoli cells to determine germ cell life or death.