DNA Synthesis In Stages Research Articles

Estrogen Receptor β Selective Ligand 5α-Androstane-3β, 17β-Diol Stimulates Spermatogonial Deoxyribonucleic Acid Synthesis in Rat Seminiferous Epithelium in Vitro

Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.

Germ cell mutagenicity of three metabolites of 1,3-butadiene in the rat: induction of spermatid micronuclei by butadiene mono-, di-, and diolepoxides in vivo.

Three metabolites of the industrial chemical 1,3-butadiene (BD), namely butadiene monoepoxide (BMO, 3,4-epoxy-1-butene), diepoxide (DEB, 1,2;3,4-diepoxybutane), and diolepoxide (DE, 3,4- epoxybutane-1,2-diol) were studied for germ cell mutagenicity using the rat spermatid micronucleus (MN) test. All three epoxides increased slightly, but significantly, the frequency of spermatid MN. The most sensitive stage to the action of BMO and DEB was preleptotene (meiotic S phase) harvested at 18-day time intervals after treatment. The dose-response for BMO followed a second order curve at this time interval, with maximum MN induction at the dose of 186 mumol/kg and lower induction of higher doses. Late stages of the meiotic prophase (late pachytene-diplotene-diakinesis) also showed some sensitivity to the three epoxides. Stem cell spermatogonia were affected by DEB as observed by a slight induction of spermatid micronuclei 50 days after treatment. No clear cytotoxic effects were observed by measuring testicular weight or cell numbers of seminiferous epithelial stage 1 18 days after the treatments. DEB at the dose 387 mumol/kg caused a slight inhibition of spermatogonial DNA synthesis in stage I and a delay of meiotic DNA replication observed in stage XII 72 hr after treatment. Since BMO is able to induce spermatid MN in the rat, the present results, together with previous data, indicate that rat bone marrow MN results that are negative for both BD and BMO cannot directly predict mutagenicity in male germ cells. The results also emphasize that tissue; species, and strain-specific differences in metabolism have to be taken into account when the genetic risks of human butadiene exposure are evaluated. The results support the conclusion that 1,3-butadiene is a germ cell mutagen-possibly also in humans.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Insulin-like growth factors selectively stimulate spermatogonial, but not meiotic, deoxyribonucleic acid synthesis during rat spermatogenesis.

The in vitro effects of insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), truncated IGF-I, insulin, and human GH (hGH) on premitotic and premeiotic DNA synthesis of adult rat germ cells in vitro were investigated. Two-millimeter segments of seminiferous tubules from four different stages containing type A4-spermatogonia (stage I), type B spermatogonia (stage V), resting preleptotene spermatocytes (stage VIIa), and preleptotene spermatocytes in the S-phase (stage VIII-IX), respectively, were isolated by transillumination-assisted microdissection. They were cultured in serum-free medium at 34 or 37 C with and without growth factors, labeled for 4 h with tritiated thymidine, and harvested at 24, 48, and 72 h. Spontaneous progression of spermatogenesis was noted at both incubation temperatures, with a more rapid rate at 37 C. IGF-I significantly stimulated [3H]thymidine uptake in originally stage I and stage V tubule segments (type A4 and B spermatogonia, respectively) after 48 h of culture at 37 C. Improved maintenance of the DNA synthesis of stage VIII-IX tubules was found after 48 h at 37 C and 72 h at 34 C. Truncated IGF-I produced a similar response, but was more potent. IGF-II showed slight stimulation of stage V tubules after 72 h at both 34 and 37 C and maintenance of stage VIII-IX tubules after 48 h at 37 C and 72 h at 34 C. hGH was effective only at 34 C, showing slight stimulation of stage I tubule segments after 48 and 72 h of incubation. Insulin at high concentrations was effective only at 37 C and stimulated DNA synthesis in stages I, V, and VIIa after 48 h and stages V and VIIa after 72 h of incubation. It is concluded that IGFs stimulate premitotic DNA synthesis of rat germ cells in vitro and may also maintain premeiotic DNA synthesis. Whether the slight response to hGH is mediated via local production of IGF-I by the tissue cultures remains to be investigated. As IGF-I and IGF-II are locally produced in the testis, the present results suggest that these factors have a selective paracrine or autocrine role in the regulation of spermatogonial proliferation during spermatogenesis.

Transforming growth factor-β modulates proliferation and differentiation of mouse clonal osteoblastic MC3T3-E1 cells depending on their maturation stages

The effect of transforming growth factor-β (TGF-β) on proliferation and differentiation of mouse clonal osteoblastic cells (MC3T3-E1) was examined in vitro in three different stages of their differentiation. Stage I (1–3 days after plating) was characterized by rapid cell growth, negligible alkaline phosphatase (ALP) activity and high proteoglycan synthesis, but low collagen production. In stage II (3–5 days after plating), proteoglycan synthesis sharply decreased and ALP activity and collagen synthesis began to increase. Stage III (7–9 days after plating) was characterized by maximal osteoblastic phenotypes. Treating MC3T3-E1 cells with 1 ng/ml of TGF-β greatly inhibited DNA synthesis in stage I but not in stage II. In contrast, TGF-β dose-dependently stimulated the synthesis of collagenase digestible proteins (CDP), noncollagenous proteins (NCP) and proteoglycan, especially in stage II. The minimum effective dose of TGF-β in this stage was as low as 0.04–0.2 ng/ml. In stages I and III, the MC3T3-E1 cells were rather insensitive to TGF-β in increasing three osteoblastic phenotypes. The increase in ALP activity in stages II and III was inhibited by 1 ng/ml of TGF-β. These results indicate that the response to TGF-β of mouse clonal osteoblastic MC3T3-E1 cells changes depending on their maturation stages.

In VitroEffects of Follicle-Stimulating Hormone, Luteinizing Hormone, and Prolactin on Follicular Deoxyribonucleic Acid Synthesis in the Hamster*

Follicles were dissected by hand or enzymatically from the ovary of the proestrous hamster at 0900 h and classified into 10 stages: stages 1-4, follicles with 1-4 layers of granulosa cells and no theca; stages 5-8, preantral follicles with 5 or more layers of granulosa cells and theca to small antral follicles; stage 9, intermediate-sized atretic antral follicles; and stage 10, healthy preovulatory antral follicles. Follicles were then incubated for 2 h with [3H]thymidine [( 3H]Tdr) in the absence or presence of gonadotropins and with incorporation of radionuclide into DNA as the end point. FSH (25 ng) significantly stimulated [3H]Tdr incorporation in all stages of follicular development with a latency of 2 h, and this effect was inhibited by 2 micrograms unlabeled Tdr. While FSH and PRL (25 and 100 ng) stimulated [3H]Tdr incorporation in all stages, LH (0.2-5 ng) action began from stage 5 onward, when definitive thecal cells and LH receptors started appearing. LH (5 ng) also suppressed 25 ng FSH-induced DNA synthesis in stages 5-10; however, stages 1-4 were unaffected. Significant increases in both intra- and extracellular cAMP levels occurred in follicles at stages 2-10 after FSH administration. In contrast, LH was active in stages 5-10, whereas PRL was ineffective. Follicular DNA synthesis increased markedly when stimulated by 8-bromo-cAMP (0.01-2 mM). These results show that gonadotropins act directly as a primary stimulus at the level of small primary and secondary follicles to regulate DNA synthesis and, thus, perhaps the growth and differentiation of granulosa and thecal cells; cAMP functions as one of the possible intracellular mediators of gonadotropin action in initiating DNA replication.