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

The Possible Role of Resource Requirements and Academic Career-Choice Risk on Gender Differences in Publication Rate and Impact

Many studies demonstrate that there is still a significant gender bias, especially at higher career levels, in many areas including science, technology, engineering, and mathematics (STEM). We investigated field-dependent, gender-specific effects of the selective pressures individuals experience as they pursue a career in academia within seven STEM disciplines. We built a unique database that comprises 437,787 publications authored by 4,292 faculty members at top United States research universities. Our analyses reveal that gender differences in publication rate and impact are discipline-specific. Our results also support two hypotheses. First, the widely-reported lower publication rates of female faculty are correlated with the amount of research resources typically needed in the discipline considered, and thus may be explained by the lower level of institutional support historically received by females. Second, in disciplines where pursuing an academic position incurs greater career risk, female faculty tend to have a greater fraction of higher impact publications than males. Our findings have significant, field-specific, policy implications for achieving diversity at the faculty level within the STEM disciplines.Comment: 9 figures and 3 table

Fibrin-Mediated Delivery of an Ovarian Follicle Pool in a Mouse Model of Infertility

The cryopreservation and autotransplantation of ovarian tissue is emerging as a powerful approach for preserving fertility. However, for cancer patients, it may not be possible to transplant ovarian tissue due to the risk of re-seeding disease. We investigated strategies for transplantation of individually isolated follicles to minimize the risk of re-introducing cancer cells present within the vasculature of ovarian stroma. Procedures for large-scale isolation of early-stage follicles and their encapsulation into fibrin hydrogels were developed. For in vivo validation studies, mice were ovariectomized and transplanted with encapsulated follicles into the ovarian bursa. A substantial increase in the number of secondary follicles was observed in the graft at 9 days after transplantation, and antral follicles by day 21, demonstrating primordial follicle recruitment into the growing pool. Initially, elevated follicle-stimulating hormone levels declined substantially by day 21, indicating feedback from the graft; presence of corpora lutea showed the graft's capability of restoring hormone cyclicity. Taken together, the transplanted follicles were able to engraft, mature, and restore ovarian function in an infertile mouse. This biomaterial may, thus, provide a platform for follicle transplantation with a low risk of cancer contamination and for developing strategies that preserve fertility for women facing a cancer diagnosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140230/1/ten.tea.2013.0675.pd

Rational Design, Synthesis, and Biological Evaluation of Progesterone-Modified MRI Contrast Agents

SummaryA series of contrast agents for magnetic resonance imaging (MRI) aimed at noninvasively determining the hormone receptor status of cancer in vitro was developed. These MRI contrast agents were prepared by conjugating progesterone to clinically used Gd(III) chelates. These agents exhibited higher progesterone receptor binding affinities in the nanomolar range and intracellular accumulation. High logP values of the modified compounds suggested that the lipophilicity of the steroid conjugates may have contributed to membrane permeability. Synchrotron radiation X-ray fluorescence microscopy and magnetic resonance images revealed that the synthesized conjugates showed the greatest cellular accumulation and significant increase in relaxivity in vitro compared to the previously developed steroid-modified agent. Transcriptional assays using the progesterone response element linked to luciferase indicated that the contrast agents entered the cell, interacted with the biological target, and drove specific progesterone-mediated transcription

A Steroid-Conjugated Contrast Agent for Magnetic Resonance Imaging of Cell Signaling

We have synthesized the first steroid hormone−MR contrast agent conjugate designed to track the cell signaling process upon binding to a gene switch system. The derivative has a high relaxivity and when tested in vitro is active as a progesterone antagonist (RU-486). By combining a transcriptional system and a noninvasive imaging technology, such as MRI, it would be a powerful tool to research the cell signaling pathway in vivo

Smad3 Mediates Activin-Induced Transcription of Follicle-Stimulating Hormone β-Subunit Gene

Synthesis of FSH by the anterior pituitary is regulated by activin, a member of the TGFβ superfamily of ligands. Activin signals through a pathway that involves the activation of the transcriptional coregulators Smad2 and Smad3. Previous work from our laboratory demonstrated that Smad3, and not Smad2, is sufficient for stimulation of the rat FSHβ promoter in a pituitary-derived cell line LβT2. Here, we used RNA interference technology to independently decrease the expression of Smad proteins in LβT2 cells to further investigate Smad2 and Smad3 roles in activin-dependent regulation of the FSHβ promoter. Down-regulation of Smad2 protein by small interfering RNA duplexes affects only basal transcription of FSHβ, whereas decreased expression of Smad3 abrogates activin-mediated stimulation of FSHβ transcription. Although highly related, Smad2 and Smad3 differ in their Mad homolog (MH) 1 domains, where the Smad2 protein contains two additional stretches of amino acids that prevent this factor from binding to DNA. We investigated whether these structural features contribute to differential FSHβ transactivation by Smad2 and Smad3. A variety of Smad chimera constructs were generated and used in transient transfection studies to address this question. Only cotransfection of chimera constructs that contain the MH1 domain of Smad3 results in activin-mediated stimulation of the rat FSHβ promoter. Furthermore, the insertion of Smad2 loops into Smad3 protein renders it inactive, suggesting that DNA binding is necessary for Smad3-mediated stimulation of the rat FSHβ promoter. Taken together, these results indicate that the functional differences between Smad2 and Smad3 in their ability to transactivate the rat FSHβ promoter lie primarily within the MH1 domain and involve structural motifs that affect DNA binding

Determining If Sex Bias Exists in Human Surgical Clinical Research

Sex is a variable that is poorly controlled for in clinical research

Role of PCSK5 Expression in Mouse Ovarian Follicle Development: Identification of the Inhibin α- and β-Subunits as Candidate Substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and βB-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability

Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin α-Subunit, a Unique TGFβ Superfamily Antagonist

Transforming growth factor-beta (TGFβ) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFβ superfamily that share a common β-subunit. The evolution of the inhibin α-subunit created the only antagonist within the TGFβ superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin α-subunit appear to reflect specific insertion-deletion changes within the inhibin β-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFβ superfamily