James K. Pru

Pregnancy and Interferon-τ Upregulate Gene Expression of Members of the 1-8 Family in the Bovine Uterus

Abstract The 1-8 family (1-8U, 1-8D, Leu-13/9-27) of interferon (IFN)-inducible genes encodes proteins that are components of multimeric complexes involved with transduction of antiproliferative and homotypic adhesion signals. Human 1-8 family members are highly similar and are regulated by type 1 and type 2 IFNs. Because the bovine uterus is bathed in conceptus-derived IFNτ during early pregnancy, it was hypothesized that members of the 1-8 family were upregulated in the bovine uterus during early pregnancy. Oligonucleotide primers were designed based on human and rat 1-8U and Leu-13 cDNAs and used in reverse transcription polymerase chain reactions to amplify bovine cDNAs from endometrial RNA. The bovine 1-8U cDNA was sequenced, found to be 84% identical to the human 1-8U, and used to screen a bovine endometrial cDNA library to isolate the full-length 1-8U and Leu-13 cDNAs. The inferred amino acid sequences of bovine 1-8U and Leu-13 were 72% and 73% identical to their respective human counterparts. Bovine 1-8U and Leu-13 retain an amino acid motif that is conserved in other 1-8 family members and in some ubiquitin-conjugating enzymes (E2s). This motif is critical for function of E2s in covalently linking ubiquitin to targeted proteins. Northern blotting revealed that bovine endometrial 1-8U and Leu-13 mRNAs were upregulated on Day 15 of pregnancy (P < 0.0001) and continued to accumulate through Day 18 of pregnancy (P < 0.05) when compared with endometrium from nonpregnant cows. The bovine 1-8U and Leu-13 mRNAs were also upregulated (P < 0.05) by IFNτ (25 nM) within 3 h, continued to accumulate through 12 h, and reached a plateau at 12–24 h in cultured bovine endometrial cells. In situ hybridization revealed that mRNAs encoding 1-8 family members were heavily localized to glandular epithelium but also were present to a lesser extent in the luminal epithelium and stroma. The temporal upregulation of 1-8U and Leu-13 mRNAs by pregnancy and IFNτ and tissue distribution of these mRNAs paralleled closely that of the ubiquitin homolog, IFN-stimulated gene product 17. These IFN-induced proteins probably work together to prepare the endometrium for adhesion of the developing conceptus.

Interferon-Tau Suppresses Prostaglandin F2α Secretion Independently of the Mitogen-Activated Protein Kinase and Nuclear Factor κ B Pathways

Abstract Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-τ on the release of prostaglandin F2α (PGF), which allows the corpus luteum to survive and continue to produce progesterone. Experiments were designed to 1) delineate the signal transduction pathway coordinating the synthesis of PGF, 2) determine how rapidly recombinant bovine (rb) IFN-τ attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-τ attenuates the secretion of PGF in cultured bovine endometrial (BEND) cells. BEND cells were untreated (control) or treated for 5, 10, 60, 180, or 300 min with PDBu (100 ng/ml), rbIFN-τ (50 or 500 ng/ml), PDBu + rbIFN-τ, or PDBu + PD98059 (MEK-1 inhibitor; 50 μM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-τ (P < 0.0001) and was ablated completely by PD98059. Parallel results were obtained for cyclooxygenase (COX)-2 protein expression. PDBu induced (P < 0.05) activation of the Raf-1/MEK-1/ERK-1/2 pathway, which was obligatory for the expression of COX-2 and secretion of PGF but was not altered by cotreatment with rbIFN-τ. PDBu induced (P < 0.05) transcription of c-jun and c-fos mRNAs within 30 min; induction was inhibited (P < 0.05) by cotreatment with PD98059 but not by cotreatment with rbIFN-τ. Treatment of BEND cells with rbIFN-τ also did not attenuate PDBu-induced degradation of IκBα, suggesting that the IκBα/NFκB pathway is not a site of IFN-τ inhibition of PGF. However, rbIFN-τ did block transcription of the COX-2 gene induced by PDBu within 30 min. In conclusion, COX-2 expression and PGF secretion induced by PDBu is mediated through the Raf-1/MEK-1/ERK-1/2 pathway, but this pathway is not disrupted by rbIFN-τ. Because rbIFN-τ inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-τ rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.

Apoptosis in Ovarian Development, Function, and Failure

Apoptosis, a form of cell death responsible for eliminating cells from the body that are damaged, senescent, potentially harmful, or no longer useful, plays a significant role in almost all aspects of ovarian development and function in vertebrate and invertebrate species. Through the years, studies of laboratory animal models and human tissues have begun to assimilate a molecular blueprint of the hormones, genes, and pathways that modulate apoptosis in ovarian germ cells and somatic cells, leading to the prospects of developing new treatments for improving reproductive health and fertility in women. In addition, significant progress has been made in elucidating a central, and perhaps somewhat surprising, role for the physiological cell death program of apoptosis in mediating pathological ovarian damage caused by exposure of women to a host of environmental, occupational, and clinical insults. However, a considerable number of the observations published to date with respect to the regulation of apoptosis in the ovary are based on correlative gene expression analyses or on the provision of single agents to isolated ovarian cells or follicles cultured in vitro. We know comparatively little of the hormones, genes, and pathways that are functionally relevant to the modulation of ovarian cell death in vivo, or of how these hormones, genes, and pathways interact to coordinate things such as granulosa cell apoptosis during follicle atresia and oocyte depletion during fetal and postnatal life. These concepts and a broad overview of the apoptosis program and its control are presented in this chapter.

Complementary deoxyribonucleic acid sequence encoding bovine ubiquitin cross-reactive protein : A comparison with ubiquitin and a 15-kDa ubiquitin homolog.

Pregnancy in the cow depends on secretion of interferon-tau (IFN-τ) by the conceptus (trophoblast and embryo) and the actions of this cytokine on the uterine endometrium. A novel 17-kDa uterine protein that is regulated by IFN-τ during early pregnancy and crossreacts with ubiquitin antiserum on Western blots, has been named bovine ubiquitin cross-reactive protein (bUCRP). We suspected that bUCRP might be structurally related to ubiquitin, and to a human UCRP (ISG15 product) that has been described in several cell lines to be regulated by Type I IFNs. In this study, immunoscreening of a bovine endometrial cDNA library with ubiquitin antiserum resulted in the isolation of cDNAs encoding bUCRP. Nucleotide sequence of the bUCRP cDNA shared 70% identity with hUCRP and 30% identity with a tandem ubiquitin repeat. Computer translation revealed that bUCRP shared the Leu-Arg-Gly-Gly (LRGG) C-terminal sequence with ubiquitin and hUCRP that has been implicated in the modulation of intracellular proteins. However, some ubiquitin residues known to function in the ligation (Arg-54) to targeted proteins and in the processing of conjugates through the proteasome (His-68), have been lost through mutation in bUCRP. Lys-48, known to function in formation of ubiquitin polymers, was present in hUCRP, but mutated to Arg in bUCRP. Because bUCRP is secreted and retains the LRGG sequence, it may have both intracellular and secreted endocrine roles in maintaining pregnancy. Bovine UCRP also may have very different intracellular roles when compared with ubiquitin and hUCRP because of mutations in residues known to form polymers and to target proteins to degradation.

Production, Purification, and Carboxy-Terminal Sequencing of Bioactive Recombinant Bovine Interferon-Stimulated Gene Product 17

Abstract An interferon (IFN)-stimulated gene (ISG) encodes a bovine 17-kDa protein (bISG17) that is released from endometrial cells but also conjugates to intracellular proteins through a ubiquitinlike mechanism. During early pregnancy in ruminants, conceptus-derived IFN-τ induces endometrial ISG17. The present experiments were designed to generate bioactive recombinant (r) bISG17. The Pichia pastoris yeast expression system was used because previous experiments expressing the human ISG15 ortholog in bacteria were confounded by inherent carboxypeptidase activity that cleaved C-terminal residues resulting in an inactive protein. In a series of extensive yeast culture experiments using shaker-bath and fermentation approaches, optimal conditions were determined for a transformant containing a multi-ISG17 gene insertion. Recombinant bISG17 was purified. Carboxy-terminal sequencing revealed that rbISG17 retained the C-terminal Gly that is potentially critical for the first step in covalent attachment to targeted intracellular proteins. The rISG17 induced (P < 0.0001) IFN-γ mRNA (reverse transcription–polymerase chain reaction) and release of IFN-γ protein (ELISA) by bovine peripheral blood mononuclear cells. The IFN-γ mRNA also was upregulated (P < 0.0001) in endometrium from pregnant (Day 18) when compared with nonpregnant (Days 14 and 18) cows. It is concluded that rbISG17 generated in a yeast expression system retains cytokine/hormonal activity. This is the first description coupling the biology of two distinct IFNs (γ and τ) through the intermediary ubiquitin homolog ISG17.