Testin is a testosterone-responsive Sertoli cell secretory product. In the present study, we demonstrated that the amount of testin secreted by Sertoli cells in vitro was comparable with several other Sertoli cell secretory products. However, virtually no testin was found in the luminal fluid and cytosols of the testis and epididymis when the intercellular junctions were not previously disrupted, suggesting that secreted testin may be reabsorbed by testicular cells in vivo. Studies using Sertoli cells with and without a cell surface crosslinker and radioiodination in conjunction with immunoprecipitation illustrated the presence of two polypeptides of 28 and 45 kDa, which constitute a binding protein complex that anchors testin onto the cell surface. The 28-and 45-kDa peptide appear to be residing on and inside the cell surface, respectively. Immunogold EM studies illustrated testin was abundantly localized on the Sertoli cell side of the ectoplasmic specialization (a modified adherens junction) surrounding developing spermatids. In contrast, very few testin gold particles were found at the site of inter-Sertoli tight junctions. When the inter-Sertoli tight junctions were formed or disrupted, no significant change in testin expression was noted. This is in sharp contrast to the disruption of Sertoli-germ cell junctions, which is accompanied by a surge in testin expression. These results demonstrate the usefulness of testin in examining Sertoli-germ cell interactions.
Previous studies established that in the rat, the uterus can accept a developing blastocyst for implantation only during a limited period of time on day 5 of gestation, termed the receptive phase. Our previous studies showed that the expression of calcitonin, a peptide hormone that regulates calcium homeostasis, is induced in rat uterus between days 3-5 of gestation and is switched off once the implantation process has progressed to day 6. In the present study, we analyze in detail how the expression of calcitonin messenger RNA (mRNA) in the uterus is regulated by the steroid hormones progesterone and estrogen and explore the possibility that calcitonin may serve as a potential marker of uterine receptivity. We demonstrate by in situ hybridization that calcitonin mRNA is synthesized specifically in the glandular epithelial cells between days 3-5 of pregnancy. Interestingly, calcitonin synthesis is also induced in these cells during pseudopregnancy, indicating that this peptide hormone is produced in the endometrium in response to maternal, rather than embryonic, signals. We also demonstrate that calcitonin mRNA expression during pseudopregnancy, like that in normal pregnancy, is under progesterone regulation. We further examined the steroid hormone regulation of uterine calcitonin expression in a delayed implantation model. In pregnant rats in which implantation is blocked upon removal of both ovaries on day 4 of gestation, continued administration of progesterone sustains calcitonin expression in the uterus for several days in the absence of estrogen. Administration of estrogen, which allows delayed implantation, also rapidly reduces calcitonin expression, indicating a role for this steroid hormone in turning off calcitonin gene expression. In gene transfection studies, expression of the progesterone receptor B isoform in cultured endometrial cells induces RNA synthesis from a reporter gene containing a 1.3-kb calcitonin promoter fragment in a hormone-dependent manner. As expected, mifepristone-complexed progesterone receptor B isoform fails to activate the calcitonin promoter. Progesterone acting through its nuclear receptor therefore regulates the expression of the calcitonin gene at the level of transcription. Finally, using RIA we investigated whether calcitonin is secreted from its glandular site of synthesis at the time of implantation by analyzing uterine flushings obtained from pregnant rats. We report the detection of a significant amount of calcitonin in the luminal secretions collected on day 4 and a lower amount on day 5 of gestation, whereas similar samples collected from animals on either day 3 or 6 of gestation did not contain detectable amounts of this peptide hormone. A transient burst of calcitonin secretion into the uterine lumen therefore occurs immediately preceding implantation. Based on these results, we propose that calcitonin is a measurable marker that forecasts the receptive state of rat endometrium during blastocyst implantation.
The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The DNA-dependent protein kinase (DNA ± PK) and the ataxia telangiectasia mutated (ATM) gene product, e ectors in the DNA damage response, contribute to the induction of c-Abl activity. The present study demonstrates that c-Abl is expressed in mouse and rat testes, and predominantly in pachytene spermatocytes of meiosis I. The results also demonstrate that c-Abl interacts directly with meiotic chromosomes. In concert with a requirement for c-Abl at the pachytene stage, we show that, in contrast to wild-type mice, testes from Abl 7/7 mice exhibit defects in spermatogenesis. These ®ndings provide the ®rst demonstration that c-Abl plays a functional role in meiosis.
Implantation of the mammalian embryo into the wall of the uterus is regulated by a timely interplay of the ovarian hormones, estrogen and progesterone. These hormones orchestrate a set of modifications in the uterine endometrium that transforms it from a nonreceptive to a receptive phase allowing the implantation of the developing blastocyst. The molecular and cellular mechanisms underlying this complex process, however, remain largely unknown. To investigate the endocrine basis of uterine receptivity, we employed a gene expression screen technique to identify factors whose expressions are modulated in the rat uterus in response to estrogen and progesterone at the onset of implantation. Here we report that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is markedly enhanced in the uterus during pregnancy. By Northern blot analysis, we show that the synthesis of calcitonin messenger RNA is induced at the time of implantation. Immunocytochemistry with calcitonin antibody demonstrates further that the peptide is localized in the glandular epithelial cells of the uterus. The antiprogestin drug RU486, which is known to block implantation, abolishes calcitonin expression, suggesting a regulatory role for progesterone in this process. Consistent with this observation, progesterone significantly stimulates calcitonin messenger RNA and protein synthesis in the uteri of ovariectomized animals. Our study, therefore, identifies calcitonin as a stage- and cell-specific marker of progesterone action in the uterus during pregnancy. Estrogen exhibits no significant effect on calcitonin expression when administered alone to ovariectomized animals. However, a low dose of estrogen synergizes with progesterone, and a high dose antagonizes progesterone-mediated gene induction. Both estrogen and progesterone, therefore, modulate calcitonin gene expression in the uterus. The stage-specific regulation of calcitonin is apparently determined by the relative concentrations and the sequences of appearance of these two hormones and possibly other as yet unknown regulatory factors during pregnancy. We propose that calcitonin, a known regulator of calcium levels in the bone and kidney, may play an important regulatory role in the uterus of pregnant animals during the early events leading to implantation of the embryo.
Testosterone produced by Leydig cells is critical for the maintenance of spermatogenesis by Sertoli cells throughout adulthood in the rat. However, the presence of androgen receptors (AR) in Leydig cells in prepubertal rats suggests additional roles for androgen in early Leydig cell function and differentiation. In the present study, AR messenger RNA (mRNA) was directly measured by in situ hybridization in sections of rat testes at three developmental stages: on day 21 postpartum, when Leydig cells exist as mesenchymal-like progenitors; on day 35, when they are still immature, producing low amounts of testosterone; and on day 90, when they are fully functional in the sexually mature animal. Testicular AR mRNA was detected in Leydig cells, pericytes, peritubular myoid cells, and Sertoli cells. On day 90, AR mRNA levels in Sertoli cells varied with the cycle of the seminiferous epithelium, achieving peak intensity at stages VII-VIII. Measurements were made by image analysis and expressed as integrated signal intensities per unit labeled area (mean +/- SEM; n = 3 rats at each age). The results showed that levels of Leydig cell and Sertoli cell AR mRNA change significantly during development (P < 0.05). Leydig cell AR mRNA was intermediate on day 21 (at 17.3 +/- 0.7), highest on day 35 (at 26.9 +/- 1.6), and lowest on day 90 (at 11.8 +/- 1.1). The trend for isolated Leydig cells from these three ages was identical. In contrast, Sertoli cell AR mRNA was lowest on day 21 (at 19.3 +/- 1.0), intermediate on day 35 (at 24.5 +/- 1.4), and highest on day 90 (at 36.9 +/- 0.5). In Leydig cells, the highest level of AR mRNA was present during puberty, whereas the greatest amount of AR mRNA in Sertoli cells was present on day 90. This indicates that Leydig cells and Sertoli cells use different mechanisms to maintain AR levels. We infer from these data that Leydig cells are maximally sensitive to androgen during puberty, which is consistent with our hypothesis that androgens facilitate their differentiation.
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