Nutritionally induced tanycytic plasticity in the hypothalamus of adult ewes.

The blood-brain barrier regulates the transport of molecules that convey global energetic status to the feeding circuitry within the hypothalamus. Capillaries within the median eminence (ME) and tight junctions between tanycytes lining the third ventricle (3V) are critical components of this barrier. Herein, we tested the hypothesis that altering the plane of nutrition results in the structural reorganization of tanycytes, tight junctions, and capillary structure within the medial basal hypothalamus. Proopiomelanocortin (POMC) neuronal content within the arcuate nucleus of the hypothalamus (ARC) was also assessed to test whether reduced nutritional status improved access of nutrients to the ARC, while decreasing the access of nutrients of overfed animals. Multiparous, nongestating ewes were stratified by weight and randomly assigned to dietary treatments offered for 75 d: 200% of dietary recommendations (overfed), 100% of dietary recommendations (control), or 60% of dietary recommendations (underfed). The number of POMC-expressing neurons within the ARC was increased (P ≤ 0.002) in underfed ewes. Overfeeding increased (P ≤ 0.01) tanycyte cellular process penetration and density compared with control and underfeeding as assessed using vimentin immunostaining. Immunostaining of tight junctions along the wall of the 3V did not differ (P = 0.32) between treatments. No differences were observed in capillary density (P = 0.21) or classification (P ≥ 0.47) within the ME. These results implicate that changes within the satiety center and morphology of tanycytes within the ARC occur as an adaptation to nutrient availability.

Follicle-stimulating hormone receptors expression in ovine corpora lutea during luteal phase: effect of nutritional plane and follicle-stimulating hormone treatment.

Corpus luteum (CL), a transient endocrine gland critical for reproductive cyclicity and pregnancy maintenance, is controlled by numerous regulatory factors. Although LH is widely recognized as the major regulator, other factors may also affect luteal functions. It has been demonstrated that FSH receptors (FSHR) are expressed not only in ovarian follicles but also in other tissues within the reproductive tract, including the CL. To evaluate FSHR expression in nontreated (nonsuperovulated; experiment 1) or FSH-treated (superovulated; experiment 2) sheep fed a control (C; maintenance), excess (O; 2 × C), or restricted (U; 0.6 × C) diet, CL were collected at the early, mid and/or late luteal phases (n = 5-7 per group). Protein and messenger RNA (mRNA) expression of FSHR were detected in the CL from all groups using immunohistochemistry followed by image analysis and quantitative RT-PCR, respectively. Follicle-stimulating hormone receptor was immunolocalized to steroidogenic small and large and nonsteroidogenic luteal cells. In both experiments, FSHR protein expression was not affected by stage of luteal development or diet. In experiment 1, expression of mRNA for all FSHR variants was greater (P <0.02 to 0.0003) at the late phase than mid or early luteal phase, and in experiment 2, it was greater (P < 0.001) at the mid than early luteal phase. Plane of nutrition did not affect FSHR mRNA expression. Comparison of FSH-treated with nontreated ewes demonstrated that FSH increased FSHR protein expression by 1.5- to 2-fold (P < 0.0001) in all groups, and mRNA expression by 7- to 30-fold (P < 0.001) for (1) FSHR-1 in all groups except U at the early luteal phase, (2) FSHR-2 in C, O, and U at the mid-phase, but not early luteal phase, and (3) FSHR-3 in U at the mid-luteal phase. Our data demonstrate that (1) FSHRs are expressed in ovine CL at several stages of luteal development, (2) FSHR protein expression does not change during the luteal phase and is not affected by diet, (3) FSHR mRNA expression not only depends on the stage of the estrous cycle but also not affected by diet in nonsuperovulated or superovulated ewes, and (4) in vivo FSH treatment enhanced FSHR protein and/or mRNA expression in the CL depending on diet and phase of the estrous cycle. Presence of FSHR in the CL indicates a regulatory role of FSH in luteal function in sheep. As very little is known about the possible role of FSH and FSHR in luteal functions, further studies should be undertaken to elucidate the endocrine, molecular, and cellular mechanisms of FSH effects on the CL.

The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep.

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-µmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet.

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.

Cotyledonary responses to maternal selenium and dietary restriction may influence alterations in fetal weight and fetal liver glycogen in sheep.

To examine the effects of maternal supranutritional selenium (Se) and nutrient restriction during mid and late gestation on placental characteristics and fetal liver glycogen, ewes received either adequate Se (ASe) or high Se (HSe) prior to breeding. On d 64 of gestation, ASe and HSe ewes remained at 100% of requirements (controls; CON) or were restricted (RES; 60% of requirements). On d 135 of gestation, fetal weight (P< or =0.08) was greatest in both HSe and CON ewes. Placentome number, mass, and caruncular and cotyledonary weight were not different (P> or =0.17) among treatments. Fetal mass:placental mass ratio was less (P=0.06) in RES compared to CON ewes. Compared to ASe, HSe exhibited increased (P< or =0.08) cellular proliferation and DNA concentration and decreased (P=0.07) cellular size in cotyledonary tissue. Nutritional restriction decreased (P< or =0.08) cotyledonary protein concentration and cellular size. VEGF receptor 1 (Flt) mRNA in cotyledonary tissue was greater in HSe compared with ASe ewes (P=0.06) and in RES compared with CON ewes (P=0.08). There was no effect of diet on caruncular growth variables (P> or =0.13) or on placental vascularity (P> or =0.11). Progesterone was greater (P< or =0.08) in ASe-RES ewes compared to all groups at d 90 and ASe-CON and HSe-CON at d 104. Although fetal glucose and cortisol concentrations were not affected by diet, fetal liver glycogen was greater (P=0.04) in ASe-RES compared to ASe-CON and HSe-RES ewes with HSe-CON being intermediate. Both Se and nutritional plane may impact placental function and fetal growth, as fetal weight and liver glycogen are altered despite similar placental vascularity measurements.

Effects of selenium supply and dietary restriction on maternal and fetal body weight, visceral organ mass and cellularity estimates, and jejunal vascularity in pregnant ewe lambs.

To examine effects of nutrient restriction and dietary Se on maternal and fetal visceral tissues, 36 pregnant Targhee-cross ewe lambs were allotted randomly to 1 of 4 treatments in a 2 x 2 factorial arrangement. Treatments were plane of nutrition [control, 100% of requirements vs. restricted, 60% of controls] and dietary Se [adequate Se, ASe (6 microg/kg of BW) vs. high Se, HSe (80 microg/kg of BW)] from Se-enriched yeast. Selenium treatments were initiated 21 d before breeding and dietary restriction began on d 64 of gestation. Diets contained 16% CP and 2.12 Mcal/kg of ME (DM basis) and differing amounts were fed to control and restricted groups. On d 135 +/- 5 (mean +/- range) of gestation, ewes were slaughtered and visceral tissues were harvested. There was a nutrition x Se interaction (P = 0.02) for maternal jejunal RNA:DNA; no other interactions were detected for maternal measurements. Maternal BW, stomach complex, small intestine, large intestine, liver, and kidney mass were less (P < or = 0.01) in restricted than control ewes. Lung mass (g/kg of empty BW) was greater (P = 0.09) in restricted than control ewes and for HSe compared with ASe ewes. Maternal jejunal protein content and protein:DNA were less (P < or = 0.002) in restricted than control ewes. Maternal jejunal DNA and RNA concentrations and total proliferating jejunal cells were not affected (P > or = 0.11) by treatment. Total jejunal and mucosal vascularity (mL) were less (P < or = 0.01) in restricted than control ewes. Fetuses from restricted ewes had less BW (P = 0.06), empty carcass weight (P = 0.06), crown-rump length (P = 0.03), liver (P = 0.01), pancreas (P = 0.07), perirenal fat (P = 0.02), small intestine (P = 0.007), and spleen weights (P = 0.03) compared with controls. Fetuses from HSe ewes had heavier (P < or = 0.09) BW, and empty carcass, heart, lung, spleen, total viscera, and large intestine weights compared with ASe ewes. Nutrient restriction resulted in less protein content (mg, P = 0.01) and protein:DNA (P = 0.06) in fetal jejunum. Fetal muscle DNA (nutrition by Se interaction, P = 0.04) concentration was greater (P < 0.05) in restricted ewes fed HSe compared with other treatments. Fetal muscle RNA concentration (P = 0.01) and heart RNA content (P = 0.04) were greater in HSe vs. ASe ewes. These data indicate that maternal dietary Se may alter fetal responses, as noted by greater fetal heart, lung, spleen, and BW.