Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.     

Selective failure of androgens to regulate follicle stimulating hormone beta messenger ribonucleic acid levels in the male rat

FSH beta, as well as LH beta, and alpha-subunit mRNA levels were examined in the pituitary glands of male rats after sex steroid replacement at various times (7, 28, or 90 days) after orchiectomy. Testosterone propionate, dihydrotestosterone propionate, or 17 beta-estradiol benzoate (E) were administered daily for 7 days before killing, to assess the role of different gonadal steroids on gonadotropin subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using rat FSH beta genomic DNA, and alpha and LH beta cDNAs. At all time points, alpha and LH beta mRNAs increased after gonadectomy and fell toward normal levels with either androgen or estrogen replacement. FSH beta mRNA levels increased variably postcastration: 4-fold at 7 days, 2-fold at 28 days, and 4- to 5-fold at 90 days. Although E replacement uniformly suppressed FSH beta mRNAs, neither testosterone propionate nor dihydrotestosterone propionate administration suppressed FSH beta mRNA levels at any time point after orchiectomy. These data demonstrate that there is a relative lack of negative regulation of FSH beta mRNA levels by androgens in a paradigm in which E administration results in marked negative regulation of FSH beta mRNA levels. Thus, in the male rat, estrogens negatively regulate all three gonadotropin subunit mRNA levels while androgens negative regulate LH beta and alpha-subunit but fail to suppress FSH beta mRNAs.     

Circulatory and hormonal changes induced by microgravity

The absence (or decrease) of the hydrostatic pressure during space flights (microgravity state) or simulations of weightlessness (by immersion, bed rest or head-down tilt) result in a body fluid shift and an engorgement of the central circulation where mechanoreceptors involved in plasma volume regulation are located. Their activation induces the initial (first hours) hormonal response with a decrease in plasma vasopressin, renin and aldosterone and probably an increase in a natriuretic factor (Gauer reflex). Prolonged exposure to microgravity leads to more complex and often hypothetical responses: cardiovascular deconditioning, modifications in secretion and circadian rhythms of above cited hormones. After 24 years of studies on approximately 200 astronauts our knowledge of cardiovascular and hormonal adaptation to space flight is still at the beginning.     

Effect of an n-3 Fatty Acid-Deficient Diet on the Adenosine-Dependent Melatonin Release in Cultured Rat Pineal

Abstract: We studied the effect of a diet deficient in n-3 fatty acids on the adenosine-dependent melatonin release from cultured rat pineal gland after stimulation by 5'- N -ethylcarboxamidoadenosine (NECA), an A₂ adenosine agonist. Experiments were conducted with 2-month-old rats raised on semipurified diets containing either peanut oil (n-3 deficients) or peanut plus rapeseed oil (controls). The proportion of docosahexaenoic acid (22:6 n-3) in the pineal total lipid fraction and in phosphatidylcholine and phosphatidylethanolamine was significantly decreased in n-3-deficient rats. This was compensated for partially by an increase in 22:4 n-6 and 22:5 n-6 levels. The activity of the cultured rat pineal, in terms of cyclic AMP content and N -acetylserotonin and melatonin release in the medium, was lower after stimulation by 10^-5 mol/L NECA in the group fed peanut oil than in the group fed peanut plus rapeseed oil. The increased ratio of n-6/n-3 fatty acids in pineal total lipids and the major glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) may have an important influence on the rat pineal responses. The results are discussed in the context of changes in membrane-bound proteins, including enzymes and/or receptors involved in the rat pineal gland function.     

Some blood genetic markers of selected tribes in Western Saudi Arabia

A total of 292 randomly selected subjects belonging to two indigenous Arab tribes (Harbi and Ghamid) and two immigrant tribes (Mograbi and Mowallad), residents in Western Saudi Arabia, have been tested for genetic variants of six blood groups, four serum proteins, and five red cell enzyme systems. The distribution of the polymorphic systems was different between indigenous and immigrant tribes, and the present Arab population shows a considerable degree of admixture from the surrounding countries, in particular Africa.     

Clinical significance of plasma MMP‐2 and MMP‐9 levels as biomarkers for tumor expression in breast cancer patients in Egypt

Matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9) are involved in the breakdown of extracellular matrix in normal physiological processes as well as in disease processes, such as cancer metastasis. We conducted this work to study the role of MMP-2 and MMP-9 in breast cancer by measuring their plasma concentrations before and after surgery. Also, to examine if their levels can reflect the stage of disease and prognosis. Forty-eight breast cancer patients and 13 patients with benign breast diseases were included in the study. MMP-2 and MMP-9 levels were measured by ELISA and semi-quantitative real-time PCR. MMP-2 and MMP-9 levels in plasma were determined by ELISA immediately before surgery and during 6 to 12 months after curative surgery. We observed a significant increase in the level of MMP-9 mRNA expression in breast cancer patients in comparison to their normal breast tissues and to tissues of benign breast disease. In all TNM tumor stages, the plasma levels of MMP-2 and MMP-9 were increased significantly before curative surgery in the studied patients with breast carcinoma and decreased significantly after surgery. Both MMP-2 and MMP-9 may be used as a possible marker for follow-up or as a marker that reflects the response of the disease to treatment.

     

Role of phytoestrogenic oils in alleviating osteoporosis associated with ovariectomy in rats

The objective of this study was to elucidate the effect of soybean oil (SbO) and sesame oil (SO) supplemented diets on bone biomarkers changes in OVX (ovariectomized) rats. The current data exhibited significant decrease in BMD (bone mineral density), accompanied with marked depletion in the level of Ca, P and Mg in both serum and bone of OVX rats. Also, serum estrogen, total protein, HDL-C (high density lipoprotein cholesterol), bone NO levels were decreased in OVX rats. However, a significant increase in the level of serum TL (total lipids), TC (total cholesterol), TG (triglycerides), LDL-C (low density lipoprotein cholesterol), VLDL-C (very low density lipoprotein cholesterol), urine minerals (Ca, P, Mg), as well as serum, bone and urine ALP (alkaline phosphatase) and ACP (acid phosphatase) activity were recorded in OVX rats. Further changes were also detected by the increased level of urine hydroxyproline, serum parathyroid hormone and osteocalcin, as well as urea and creatinine level in both serum and urine. On the other hand, when OVX rats were fed on SbO (soy bean oil) (15 % w/w) or SO (sesame oil) (10 % w/w) supplemented diets, the data recorded a significant improvement in all the above mentioned parameters. So, it can be concluded that consumption of SbO or SO supplemented diets might be considered as a functional food for retarding risks of osteoporosis associated with estrogen deficiency in OVX states.     

Family‐assisted inference of the genetic architecture of major histocompatibility complex variation

With their direct link to individual fitness, genes of the major histocompatibility complex (MHC) are a popular system to study the evolution of adaptive genetic diversity. However, owing to the highly dynamic evolution of the MHC region, the isolation, characterization and genotyping of MHC genes remain a major challenge. While high‐throughput sequencing technologies now provide unprecedented resolution of the high allelic diversity observed at the MHC, in many species, it remains unclear (i) how alleles are distributed among MHC loci, (ii) whether MHC loci are linked or segregate independently and (iii) how much copy number variation (CNV) can be observed for MHC genes in natural populations. Here, we show that the study of allele segregation patterns within families can provide significant insights in this context. We sequenced two MHC class I (MHC‐I) loci in 1267 European barn owls (Tyto alba), including 590 offspring from 130 families using Illumina MiSeq technology. Coupled with a high per‐individual sequencing coverage (~3000×), the study of allele segregation patterns within families provided information on three aspects of the architecture of MHC‐I variation in barn owls: (i) extensive sharing of alleles among loci, (ii) strong linkage of MHC‐I loci indicating tandem architecture and (iii) the presence of CNV in the barn owl MHC‐I. We conclude that the additional information that can be gained from high‐coverage amplicon sequencing by investigating allele segregation patterns in families not only helps improving the accuracy of MHC genotyping, but also contributes towards enhanced analyses in the context of MHC evolutionary ecology.