Histone methylation is a critical regulator of the abnormal expression of POU5F1 and RASSF1A in testis cancer cell lines.

DNA and histone methylation are epigenetic modifications functioning in transcriptional control and have been implicated in the deregulation of gene expression in cancer. As a first step to determine if histone methylation could be involved in testis cancer pathogenesis, we performed immunofluorescent localization of histone H3 methylation at lysine 4 (H3-K4; gene activating) and lysine 9 (H3-K9; gene silencing) in healthy testis tissue and in samples of non-seminoma germ-cell tumours. In healthy testis, the distribution of histone H3 methylation was dependent on the developmental stage of spermatogenic cells and in non-seminoma, histone H3-K4 and K9 methylation was detected in all histological subtypes. This suggested that histone H3-K4 and K9 methylation could be associated with abnormal gene expression in non-seminoma. To determine the gene-specific function of histone H3 methylation, we proceeded to define the epigenetic status of key genes implicated in the pathogenesis of non-seminoma, namely the proto-oncogene POU5F1, which is overexpressed in testis cancer, and the tumour suppressor RASSF1A, which is aberrantly silenced. Cell lines representative of non-seminoma were treated with the chromatin-modifying drug, 5-aza-2'-deoxycytidine (5-aza-dC). Chromatin immunoprecipitation and real-time polymerase chain reaction analyses revealed that treatment with 5-aza-dC restored RASSF1A expression through a loss of gene silencing H3-K9 methylation and by retention of gene activating H3-K4 tri-methylation in the promoter region. In contrast, the expression of POU5F1 was reduced by 5-aza-dC and was associated with a loss of gene activating H3-K4 di-methylation in the promoter region. Analysis of DNA methylation revealed a slight reduction in DNA hypermethylation at the RASSF1A promoter, whereas the POU5F1 promoter remained mostly unmethylated and unaffected. Our results indicate that the effects of 5-aza-dC on histone methylation profiles are gene-specific and that aberrant histone modifications may serve as a principal means of misregulation of RASSF1A and POU5F1 expression in testis cancer.

Folic acid supplementation reduces multigenerational sperm miRNA perturbation induced by in utero environmental contaminant exposure.

Persistent organic pollutants (POPs) can induce epigenetic changes in the paternal germline. Here, we report that folic acid (FA) supplementation mitigates sperm miRNA profiles transgenerationally following in utero paternal exposure to POPs in a rat model. Pregnant founder dams were exposed to an environmentally relevant POPs mixture (or corn oil) ± FA supplementation and subsequent F1-F4 male descendants were not exposed to POPs and were fed the FA control diet. Sperm miRNA profiles of intergenerational (F1, F2) and transgenerational (F3, F4) lineages were investigated using miRNA deep sequencing. Across the F1-F4 generations, sperm miRNA profiles were less perturbed with POPs+FA compared to sperm from descendants of dams treated with POPs alone. POPs exposure consistently led to alteration of three sperm miRNAs across two generations, and similarly one sperm miRNA due to POPs+FA; which was in common with one POPs intergenerationally altered sperm miRNA. The sperm miRNAs that were affected by POPs alone are known to target genes involved in mammary gland and embryonic organ development in F1, sex differentiation and reproductive system development in F2 and cognition and brain development in F3. When the POPs treatment was combined with FA supplementation, however, these same miRNA-targeted gene pathways were perturbed to a lesser extend and only in F1 sperm. These findings suggest that FA partially mitigates the effect of POPs on paternally derived miRNA in a intergenerational manner.

Prenatal exposure to an environmentally relevant mixture of Canadian Arctic contaminants decreases male reproductive function in an aging rat model.

Elevated levels of organochlorines (OC) have been reported in Inuit populations in the Arctic. We hypothesized that prenatal exposure to a Canadian Arctic OC mixture adversely affects male reproductive function and health with age. Sprague-Dawley female rats (F0) were gavaged with an environmentally relevant concentration of an Arctic OC mixture or corn oil (Control) during mating with untreated males until parturition (F1 litters). After postnatal day (PND) 90, the weights of the OC F1 males differed dramatically relative to Controls (P<0.05; n=10) and they exhibited respiratory distress. Except for possible thinning of the alveolar barrier, histological observation of the lungs revealed no apparent pathology to explain the respiratory distress. At PND 365, OC F1 males had reduced relative reproductive organ weights and lower sperm quality than Controls (P<0.05). At PND 90, OC F1 males were subfertile (P<0.05), but were infertile at PND 365. In conclusion, environmentally relevant prenatal OC exposure reduced reproductive function and health in aging male rats, providing new insight into the effects of early-life exposures to these contaminants.

Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2.

Recognition and establishment of pregnancy involve several molecular and cellular interactions among the conceptus, uterus, and corpus luteum (CL). In ruminants, interferon-tau (IFNtau) of embryonic origin is recognized as the pregnancy recognition signal. Endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin, whereas PGE(2) is considered a luteoprotective or luteotrophic mediator at the time of establishment of pregnancy. The interplay between IFNtau and endometrial PGs production, transport, and signaling at the time of maternal recognition of pregnancy (MRP) is not well understood. We have studied the expression of enzymes involved in metabolism of PGE(2) and PGF(2alpha), cyclooxygenase-1 (COX-1) and COX-2, PG synthases (PGES and PGFS), PG 15-dehydrogenase, and PG transporter as well as PGE(2) (EP2 and EP3) and PGF(2alpha) receptors. IFNtau influences cell-specific expression of COX-2, PGFS, EP2, and EP3 in endometrium, myometrium, and CL in a spatio-temporal and tissue-specific manner, whereas it does not alter COX-1, PGES, PG 15-dehydrogenase, PG transporter, or PGF(2alpha) receptor expression in any of these tissues. In endometrium, IFNtau decreases PGFS in epithelial cells and increases EP2 in stroma. In myometrium, IFNtau decreases PGFS and increases EP2 in smooth muscle cells. In CL, IFNtau increases PGES and decreases EP3. Together, our results show that IFNtau directly or indirectly increases PGE(2) biosynthesis and EP2-associated signaling in endometrium, myometrium, and CL during MRP. Thus, PGE(2) may play pivotal roles in endometrial receptivity, myometrial quiescence, and luteal maintenance, indicating polycrine (endocrine, exocrine, paracrine, and autocrine) actions of PGE(2) at the time of MRP. Therefore, the establishment of pregnancy may depend not only on inhibition of endometrial PGF(2alpha), but also on increased PGE(2) production in cattle.

Oestrous cycle and pregnancy effects on the distribution of oestrogen and progesterone receptors in bovine endometrium.

Reproductive cyclicity and pregnancy require cell and tissue specific expression of oestrogen (OR) and progesterone receptors (PR). To determine the effects of cycle day and early pregnancy (days 16-30) on the distribution of OR and PR, uteri were collected from cycling (n=39), pregnant (n=14), and steroid-treated ovariectomized cattle (n=10). Immunohistochemistry was used to localize OR and PR, and staining intensity was scored. Moderate staining for the PR was detected in the stroma at proestrus (days 17-20). Expression increased through oestrous, reaching maximal levels by metestrus (days 1-6) (P< 0.05). During diestrus (days 7-16), PR expression was downregulated (P< 0.05). Progesterone receptor expression was detected in the luminal epithelium in one of three animals each at days 3 and 6 of the oestrous cycle. In pregnant animals only, caruncular stroma showed reduced PR staining compared to intercaruncular stroma (P< 0.05). Reactivity to the OR antibody in cycling animals was detected in the stroma and glandular epithelium. Levels increased through proestrus and oestrous, were maximal during metestrus (P< 0.05), and decreased during diestrus (P< 0.05). Interestingly, there was transient upregulation in the luminal epithelium on day 16 or day 14 (P< 0.05). This was not evident at day 16 of pregnancy, although some OR reactivity was present in stroma and glands. By day 18 of pregnancy OR were undetectable (P< 0.05). Oestrogen-treated ovariectomized animals had increased OR and PR expression in comparison to progesterone treated cows (P< 0.05). Untreated ovariectomized cows showed constitutive steroid receptor expression. These results support the widely accepted role for sex steroids in the regulation of their receptors and indicate that there is paracrine regulation of steroid receptor expression in the bovine uterus.

Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins.

The selective modulation of transcription exerted by steroids depends upon recognition of signalling molecules by properly folded cytoplasmic receptors and their subsequent translocation into the nucleus. These events require a sequential and dynamic series of protein-protein interactions in order to fashion receptors that bind stably to steroids. Central to receptor maturation, therefore, are several molecular chaperones and their accessory proteins; Hsp70, Hsp40, and hip modulate the 3-dimensional conformation of steroid receptors, permitting reaction via hop with Hsp90, arguably the central protein in the process. Binding to Hsp90 leads to dissociation of some proteins from the receptor complex while others are recruited. Notably, p23 stabilizes receptors in a steroid binding state, and the immunophilins, principally CyP40 and Hsp56, arrive late in receptor complex assembly. In this review, the functions of molecular chaperones during steroid receptor maturation are explored, leading to a general mechanistic model indicative of chaperone cooperation in protein folding.

Aortic aneurysm: to tube or not to tube.

One hundred seventy-five patients underwent aortic aneurysm replacement from 1970 to 1977, and these patients were reviewed retrospectively as to the need for further aortic surgery because of progression of atherosclerosis or aneurysmal disease. A 99% (174 of 175) follow-up was obtained. Eighty-six patients had tube graft replacement operations. Selection of tube graft replacement as opposed to other operative techniques included physical examination, documentation of segmental pressures by Dopper studies, and aortography. Seventy-three percent of all patients undergoing aortic aneurysm replacement had angiography performed before surgery to demonstrate the location of the aneurysm as well as evidence of occlusive disease. Of the eighty-six patients with tube graft replacements (mean follow-up 4.86 years +/- 2 years), two patients required aortobifemoral grafting--one for iliac stenosis, the other for an occluded iliac artery secondary to a thrombosed femoral aneurysm. Only 6 of the 86 patients who underwent tube graft replacement developed symptoms of claudication. None of these patients required operation. This retrospective study demonstrates that placement of a tube graft for aortic aneurysm is a reliable operation in carefully selected patients. Few of these patients require repeat aortic surgery for occlusive or aneurysmal iliofemoral disease. Extensive follow-up of tube graft patients is discussed.

Employment following peripheral vascular surgery: an increasingly critical issue.

As discussion for decreasing social security benefits or increasing the age of retirement ensues, the effectiveness of maintaining patients' employment following vascular surgery becomes extremely important. Two hundred fifty-five major vascular operations were performed from 1977 to 1981 on an employed populace. There was a 64% incidence of return to full employment following a major vascular procedure. The ability to return to work was statistically related to the degree of success of surgery following carotid endarterectomy, aortofemoral bypass for occlusive disease, femoral-distal and femoropopliteal bypass. The ability to work following abdominal aortic aneurysm resection, however, was statistically related to the age of the patient, not the outcome of the operation. Seventy-five percent of patients whose operative indication for aortofemoral and femoropopliteal bypass was claudication returned to work. Only 3% of patients who had been receiving disability benefits for vascular disease prior to vascular reconstruction returned to work. There was no correlation with the success of the operation or the age of the patient. One of the goals involved in performing vascular surgery is to return the patient to full employment. It is encouraging to see that this goal is achieved.

A review of one hundred forty-seven popliteal aneurysms with long-term follow-up.

A 20-year experience with a collected series of 147 popliteal aneurysm in 87 patients is reviewed; there were 84 male patients. Ages ranged from 42 to 90 years with a median age of 60.2. Bilateral aneurysms were found in 60 patients (68%). Ninety-eight extremities presented with symptoms, whereas 94 aneurysms had one or more preoperative complications. Sixty-six (45%) were thrombosed, 34 (23%) had embolized, and four (3%) had ruptured. Associated aneurysms were found in 55% of the total group and in 68% of those with bilateral popliteal aneurysms. Forty percent of all patients had abdominal aortic aneurysms, whereas 34% had femoral aneurysms and 25% had iliac aneurysms. Therapy included bypass grafting (99), observations (26), primary amputation (12), sympathectomy (3), and exploration only (7). In 32 limbs, grafts became occluded during the follow-up period. All except one of the occluded grafts were in patients with preoperative symptoms related to the aneurysm, and all but one primary form of therapy and 22 as a secondary procedure. All were associated with preoperative vascular ischemia or a complicated aneurysm. Complete, detailed, long-term follow-up of 1 to 14 years is reported for 65 patients. The overall follow-up averaged 44 months. Death rates were shown by life-table analysis to be significantly greater than rates among the general population. Complications of aneurysms were very common (64%) and when the occurred, 36% ended in amputation. Therefore, elective replacement of the aneurysm at the time of diagnosis is recommended.

Prognosis of patients with transient ischemic attacks and normal angiograms.

Of 358 patients referred for evaluation of cerebral vascular insufficiency over a ten-year period (January 1971 to April 1981), 32 were isolated who had experienced hemispheric transient ischemic attacks (TIAs), and lacking other identifiable abnormality were found angiographically to be free of suspicious extracranial occlusive or ulcerated lesions. These patients were studied retrospectively to evaluate their prognosis in terms of cerebral infarction and recurrent neurologic symptoms. In a mean follow-up of 16 months, ranging from three months to four years, no patient suffered a cerebral infarction. Six had further symptoms; however three were due to other abnormalities found subsequently. Three patients continued to be symptomatic; however, only one patient had recurrent hemispheric symptoms, the others being global and vague in description. Severe hypertension was associated with an increased risk of further attacks. The data indicate that the prognosis for patients with normal angiograms and hemispheric TIAs, lacking other identifiable abnormality, is good.

Prospective randomized study of concomitant lumbar sympathectomy with aortoiliac reconstruction.

Ninety-three consecutive patients for aortofemoral bypass grafting were studied to determine the effect of sympathectomy on early and late patency, the need for subsequent distal procedures, amputation rate, and ankle-brachial Doppler systolic pressure indexes. Sixty-seven patients completed the study. Fifty-three limbs were randomized to receive sympathectomy and 81 no sympathectomy. The mean length of follow-up postoperatively was 11 +/- 2.53 months (maximum 24). Five grafts occluded, two early and three late. Both early occlusions occurred in the nonsympathectomy group and all three late occlusions ensued in the sympathectomy group. Subsequent distal procedures were necessary early (within 30 days) in five limbs, all in the nonsympathectomy group. Nine limbs required distal procedures late, four (5 percent) in the nonsympathectomy group and five (9.5 percent) in the sympathectomy group (p greater than 0.05). Altogether, distal bypass was required in 11 percent of the patients in the nonsympathectomy group versus 9.4 percent in the sympathectomy group (p greater than 0.05). Five limbs were amputated 4 to 14 months after aortofemoral bypass, four (5 percent) in the nonsympathectomy and one (2 percent) in the sympathectomy group (p greater than 0.05). The ankle-brachial pressure index increased significantly after aortofemoral bypass in both groups (p less than 0.05). The mean increase after aortofemoral bypass was more marked in the sympathectomy group (p greater than 0.05). Thus, at a mean follow-up time of 11 months after aortofemoral bypass, there was no significant difference in graft patency, need for subsequent distal bypass, or amputation rate between the sympathectomy and nonsympathectomy groups.

Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes.

Epidemiological studies suggest that a father's diet can influence offspring health. A proposed mechanism for paternal transmission of environmental information is via the sperm epigenome. The epigenome includes heritable information such as DNA methylation. We hypothesize that the dietary supply of methyl donors will alter epigenetic reprogramming in sperm. Here we feed male mice either a folate-deficient or folate-sufficient diet throughout life. Paternal folate deficiency is associated with increased birth defects in the offspring, which include craniofacial and musculoskeletal malformations. Genome-wide DNA methylation analysis and the subsequent functional analysis identify differential methylation in sperm of genes implicated in development, chronic diseases such as cancer, diabetes, autism and schizophrenia. While >300 genes are differentially expressed in offspring placenta, only two correspond to genes with differential methylation in sperm. This model suggests epigenetic transmission may involve sperm histone H3 methylation or DNA methylation and that adequate paternal dietary folate is essential for offspring health.

Cyclic modulation of integrin expression in bovine endometrium.

Integrins are heterodimeric glycoproteins involved in cell-cell and cell-extracellular matrix adhesion. In this study, the spatial and temporal distribution of selected integrins and extracellular matrix proteins was determined in bovine endometrium from cycling and ovariectomized animals using indirect immunohistochemistry. The expression of integrins alpha(6) and alpha(v)beta(3) was estrous cycle-dependent. Strong immunostaining for alpha(v)beta(3) occurred in the basement membrane region of intercaruncular luminal epithelium except on Day 16 (P<0.05). Staining of subepithelial stromal cells declined in diestrous samples (P<0.05). In all samples, there was reduced alpha(v)beta(3) reactivity in the caruncles. Staining for alpha(6) decreased in the epithelial basement membrane at proestrus through estrus (Days 18-0). Expression of integrin subunits alpha(3) and alpha(4) was cycle-independent. Moderate staining for alpha(3) was detected on epithelium and alpha(4) was present on stromal cells. The distribution of beta(1) suggested dimerization with alpha(3), alpha(4), and alpha(6). Laminin was detected in the epithelial and vasculature basement membranes. Collagen IV was present in the glandular epithelium basement membrane and subepithelial stromal cells, whereas fibronectin was found only in the stroma. Estrous cycle-dependent distribution and expression of alpha(v)beta(3) and alpha(6) suggest their regulation by ovarian steroids, growth factors, and prostaglandins.