Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

BACKGROUND: Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA. METHODS: The subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends (RACE) RT-PCR from adult rhesus monkey testis RNA. RESULTS: Both the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0), which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1). Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained. CONCLUSION: The data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action

Gonadotrophin-mediated miRNA expression in testis at onset of puberty in rhesus monkey: Predictions on regulation of thyroid hormone activity and DLK1-DIO3 locus

Molecular mechanisms responsible for the initiation of primate spermatogenesis remain poorly characterized. Previously, 48 h stimulation of the testes of three juvenile rhesus monkeys with pulsatile LH and FSH resulted in down-regulation of a cohort of genes recognized to favor spermatogonia stem cell renewal. This change in genetic landscape occurred in concert with amplification of Sertoli cell proliferation and the commitment of undifferentiated spermatogonia to differentiate. In this report, the non-protein coding small RNA transcriptomes of the same testes were characterized using RNA sequencing: 537 mature micro-RNAs (miRNAs), 322 small nucleolar RNAs (snoRNAs) and 49 small nuclear RNAs (snRNAs) were identified. Pathway analysis of the 20 most highly expressed miRNAs suggested that these transcripts contribute to limiting the proliferation of the primate Sertoli cell during juvenile development. Gonadotrophin treatment resulted in differential expression of 35 miRNAs, 12 snoRNAs and four snRNA transcripts. Ten differentially expressed miRNAs were derived from the imprinted delta-like homolog 1-iodothyronine deiodinase 3 (DLK1-DIO3) locus that is linked to stem cell fate decisions. Four gonadotrophin-regulated expressed miRNAs were predicted to trigger a local increase in thyroid hormone activity within the juvenile testis. The latter finding leads us to predict that, in primates, a gonadotrophin-induced selective increase in testicular thyroid hormone activity, together with the established increase in androgen levels, at the onset of puberty is necessary for the normal timing of Sertoli cell maturation, and therefore initiation of spermatogenesis. Further examination of this hypothesis requires that peripubertal changes in thyroid hormone activity of the testis of a representative higher primate be determined empirically.Fil: Aliberti, Paula. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Servicio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sethi, Rahil. University of Pittsburgh; Estados UnidosFil: Belgorosky, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Servicio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chandran, Uma R.. University of Pittsburgh; Estados UnidosFil: Plant, Tony M.. University of Pittsburgh; Estados UnidosFil: Walker, William H.. University of Pittsburgh; Estados Unido

The ethics of practical reasoning : exploring the terrain

Social work has been under sustained scrutiny regarding the quality of decision-making. The assumption is that social workers make poor quality decisions. And yet our knowledge and understanding of how social workers make decisions is, at best, partial. In our view, examination of practitioner decision-making will be enhanced by considering the role that ethics plays in practical judgement in practice. Although there has been significant work regarding the role of values and ethics in practice, this work tends to idealise morality setting up external standards by which practice is judged. In this paper, we will argue that ethics in practice needs to be understood as more than simply operationalise ideal standards, ethics also entails critical engagement with social and ethical issues and can challenge idealised statements of values. We outline the idea of the ethical dimension of practical reasoning, consider its relationship to professional discretion, judgments and decision-making in order to provide a clear focus for this research agenda, and identify the practical challenges researching ethics in professional decision-making entails

Atrazine and Breast Cancer: A Framework Assessment of the Toxicological and Epidemiological Evidence

The causal relationship between atrazine exposure and the occurrence of breast cancer in women was evaluated using the framework developed by Adami et al. (2011) wherein biological plausibility and epidemiological evidence were combined to conclude that a causal relationship between atrazine exposure and breast cancer is “unlikely”. Carcinogenicity studies in female Sprague-Dawley (SD) but not Fischer-344 rats indicate that high doses of atrazine caused a decreased latency and an increased incidence of combined adenocarcinoma and fibroadenoma mammary tumors. There were no effects of atrazine on any other tumor type in male or female SD or Fischer-344 rats or in three strains of mice. Seven key events that precede tumor expression in female SD rats were identified. Atrazine induces mammary tumors in aging female SD rats by suppressing the luteinizing hormone surge, thereby supporting a state of persistent estrus and prolonged exposure to endogenous estrogen and prolactin. This endocrine mode of action has low biological plausibility for women because women who undergo reproductive senescence have low rather than elevated levels of estrogen and prolactin. Four alternative modes of action (genotoxicity, estrogenicity, upregulation of aromatase gene expression or delayed mammary gland development) were considered and none could account for the tumor response in SD rats. Epidemiological studies provide no support for a causal relationship between atrazine exposure and breast cancer. This conclusion is consistent with International Agency for Research on Cancer’s classification of atrazine as “unclassifiable as to carcinogenicity” and the United States Environmental Protection Agency's classification of atrazine as “not likely to be carcinogenic.

Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta)

In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitroprimate spermatogenesis and for possible future application to man

Neuropeptide Y: A hypothalamic brake restraining the onset of puberty in primates

The adult reproductive axis is driven by an intermittent discharge of gonadotropin-releasing hormone (GnRH) generated by a network of hypothalamic neurons known as the GnRH pulse generator. Although this signal generator is operational in infant primates, puberty in these species is delayed by activation shortly after birth of a central neural mechanism that holds GnRH release in check during juvenile development. Here, we show that, in the male rhesus monkey, the postnatal pattern in GnRH pulse generator activity is inversely related to that in neuropeptide Y (NPY) gene and protein expression in the mediobasal hypothalamus and that central administration of an NPY Y1 receptor antagonist to juvenile animals elicits precocious GnRH release. Cell imaging indicated that the developmentally regulated NPY neurons may be located in regions dorsal to the arcuate nucleus. These findings lead us to propose that NPY is a fundamental component of the neurobiological brake restraining the onset of puberty in primates