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Biomedical subjects

C A Bagnell

Publications and source records attributed to C A Bagnell.

At least 19 recordsLinked to original sources
PubMed2006

Uterine development and endometrial programming.

Structural patterning and functional programming of uterine tissues are mechanistically coupled. These processes ensure anteroposterior differentiation of uterine tissues from adjacent segments of the developing female reproductive tract (FRT) and radial patterning that establishes uterine-specific histoarchitecture and functionality. Uterine organogenesis begins prenatally and is completed postnatally. Genes required for FRT development include Pax2, Lim1 and Emx2, genes in the abdominal-B Hoxa cluster, and members of both Wnt and Hedgehog (Hh) gene families. Disruption of morphoregulatory gene expression patterns can prevent FRT development entirely or compromise uterine organogenesis specifically. Oestrogen receptor-alpha (ER) -dependent events associated with development of the neonatal porcine uterus can be altered by administration of oestrogen (E) or relaxin (RLX). Expression of the RLX receptor is detectable in porcine endometrium at birth, before onset of ER expression and uterine gland genesis. Uterotrophic effects of both E and RLX can be inhibited with the ER antagonist ICl 182,780, indicating that RLX may act via crosstalk with the ER system in neonatal tissues. Exposure of neonatal gilts to E alters temporospatial patterns of Hh, Wnt and Hoxa expression in the uterine wall. Oestrogen given for two weeks from birth produced hypoplastic adult porcine uteri that were less responsive to periattachment conceptus signals as reflected by reduced growth response and luminal fluid protein accumulation, altered endometrial gene expression, and reduced capacity for conceptus support. Data reinforce the concept that factors affecting signalling events in uterine tissues that produce changes in morphoregulatory gene expression patterns during critical organisational periods can alter the developmental trajectory of the uterus with lasting consequences. Thus, uterine tissues can be programmed epigenetically for success or failure during perinatal life.

Animals↗
PubMed2001

Systemic relaxin in pregnant pony mares grazed on endophyte-infected fescue: effects of fluphenazine treatment.

Tall fescue is one of the most widely grown forage grasses for horses in the United States. However, it is frequently infected with the endophyte Neotyphodium coenophialum which produces ergot alkaloids that cause severe adverse effects in the pregnant mare. The objectives of this study were to determine the effects of fescue toxicosis and fluphenazine on circulating relaxin in pregnant pony mares and evaluate the usefulness of relaxin as a monitor of treatment efficacy. Twelve mares were maintained on endophyte-infected tall fescue pasture. Group TRT (n = 6), received 25 mg of fluphenazine decanoate (i.m.) on Day 320 of gestation while Group UTRT served as untreated controls. Daily blood samples were collected from Day 300 of gestation until Day 3 post partum and analyzed for plasma relaxin concentrations using a homologous equine radioimmunoassay. Mean gestation lengths were 330 +/- 0.7 and 336.5 +/- 3.2 days for TRT and UTRT mares, respectively (P = 0.07). Mean plasma relaxin concentrations in both groups of mares during the week before treatment (Day 313 to 319) were not different (UTRT, 53.4 +/- 11.3 ng/mL; TRT, 61.4 +/- 9.3 ng/mL). In the week after treatment (Day 320 to 326), mean plasma relaxin tended to be higher (P = 0.1) in TRT mares (66.7 +/- 6.2 ng/mL) when compared with UTRT mares (49.6 +/- 6.6 ng/mL), representing a 17.1 ng/mL difference in circulating relaxin between the two groups. Systemic relaxin during the last week before delivery (days relative to parturition) for UTRT and TRT mares was 45.7 +/- 6.7 and 64.7 +/- 6.4 ng/mL (P = 0.06), respectively. At Day -8 and Day -5 relative to parturition, systemic relaxin in TRT mares was significantly higher (P < 0.05) than in UTRT mares. Three of the six UTRT mares and one TRT mare showed clinical symptoms of fescue toxicosis. In the week before delivery, circulating relaxin in mares with problematic pregnancies (39.9 +/- 7.8 ng/mL) was significantly lower than concentrations measured in mares with normal pregnancies (63.4 +/- 5.4 ng/mL; P = 0.03). Clinical observations suggest that a one-time injection with fluphenazine improved pregnancy outcome by reducing the adverse effects of fescue toxicosis concomitant with a stabilization of plasma relaxin concentrations. These data support the hypothesis that systemic relaxin may be a useful biochemical means of monitoring placental function and treatment efficacy in the mare.

Acremonium↗
PubMed2001

Relaxin increases secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 during uterine and cervical growth and remodeling in the pig.

Matrix metalloproteinases are proteolytic enzymes that degrade the extracellular matrix and are essential for tissue remodeling. Uterine and cervical growth require remodeling of structural barriers to cell invasion and matrix metalloproteinase-2 and -9 degrade type IV collagen, the major component of basement membranes. Relaxin stimulates uterine and cervical growth and remodeling, which includes remodeling of support elements such as basement membranes. The objective of this study was to determine whether relaxin alters the production and/or activity of matrix metalloproteinase-2 and -9 in the uterus or cervix of the pig. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. The expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 was characterized by gel zymography, and proteins were quantified by immunoblotting. Total enzyme activity was measured using matrix metalloproteinase-specific fluorescent substrate assays. In both uterine and cervical tissues, immunoreactive matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression was similar in relaxin-treated and control animals. However, tissue-associated gelatinase activity was attenuated by relaxin (P < 0.05). In contrast, relaxin significantly increased the secretion of active matrix metalloproteinase-2 and -9 protein into uterine fluid (P < 0.05). Given the importance of matrix metalloproteinases in extracellular matrix degradation, the observation that relaxin promotes uterine secretion of matrix metalloproteinase-2 and -9 supports the concept that relaxin facilitates the growth and remodeling of reproductive tissues by increasing extracellular proteolysis in the pig reproductive tract.

Animals↗
PubMed2001

Expression of uterine and cervical epithelial cadherin during relaxin-induced growth in pigs.

Epithelial cadherin (E-cadherin), a member of the cadherin family of calcium-dependent adhesion molecules, is present in reproductive tissues. Relaxin, a hormone important for uterine and cervical growth in pigs, increases the expression of E-cadherin in the MCF-7 mammary epithelial cell line. The objective of this study was to characterize the expression of E-cadherin during relaxin-induced growth of the uterus and cervix in an immature pig model, independent of high circulating steroids. After administration of relaxin to prepubertal gilts, the uterus and cervix were collected. E-cadherin mRNA and protein were measured by northern and western blot analysis, respectively. A 120 kDa protein band, corresponding to E-cadherin, was detected in all tissues examined. Relaxin significantly (P < 0.05) increased the amount of E-cadherin protein in the uterus (P < 0.05), whereas no significant changes were observed in E-cadherin protein in the cervix. A 4.2 kb E-cadherin transcript was detected in all tissues and E-cadherin mRNA was significantly higher (P < 0.05) in uteri from relaxin-treated gilts compared with control gilts. E-cadherin was localized by immunocytochemistry to the epithelial cells of the uterine and cervical lumen, and the uterine glandular epithelium. Quantitative analysis revealed that administration of relaxin significantly increased (P < 0.05) the height of the uterine luminal epithelium compared with that of the controls. This is the first report of the expression of E-cadherin in the uterus and cervix of pigs. The findings from this study indicate that relaxin increases the expression of uterine E-cadherin in the reproductive tract of pigs. Administration of relaxin to prepubertal gilts in vivo increased uterine epithelial cell growth independent of circulating steroids, with a concomitant increase in E-cadherin expression.

Animals↗
PubMed2000

Inhibition of pig granulosa cell adhesion and growth in vitro by immunoneutralization of epithelial cadherin.

Epithelial cadherin (E-cadherin) is a member of the cadherin family of calcium-dependent cell adhesion molecules and is present in the ovary. Although expression of E-cadherin is high in healthy pig granulosa cells and low in granulosa cells of atretic follicles, the importance of E-cadherin-mediated adhesion in granulosa cell function is unclear. The aim of the present study was to determine the impact of immunoneutralization of E-cadherin on granulosa cell adhesion, DNA synthesis and cell proliferation in vitro. Before attachment, pig granulosa cells were exposed to a monoclonal E-cadherin antibody (DECMA-1) which blocks E-cadherin function. Controls included substitution of the antibody with either mouse ascites fluid or another E-cadherin antibody directed against the cytoplasmic domain and which was therefore inaccessible in intact cells. Both granulosa cell proliferation and insulin-like growth factor I-induced DNA synthesis were inhibited significantly in the presence of DECMA-1 compared with controls (P < 0.05). Control granulosa cells in culture formed large clusters with many cells packed tightly together. However, after 48 h exposure to the function-perturbing E-cadherin antibody, there was a significant decrease in the size of the granulosa cell clusters (P < 0.05) and the degree of cell-cell contact was reduced compared with control cultures. No effects on DNA synthesis, cell proliferation or cell adhesion were observed when DECMA-1 was substituted with either mouse ascites fluid or the antibody specific for the cytoplasmic domain of E-cadherin. In conclusion, these data provide evidence to support the hypothesis that E-cadherin is important for maintaining granulosa cell contact, DNA synthesis and cell proliferation in vitro. These results indicate that E-cadherin plays a fundamental role in maintaining both the structure and function of ovarian follicles.

Analysis of Variance↗
PubMed1999

Trophic effects of relaxin on reproductive tissue: role of the IGF system.

Although the growth promoting actions of relaxin on the reproductive tract have been well documented, the means by which relaxin stimulates reproductive tissue growth has not been identified. This report is an overview of studies from our laboratory investigating the role of the insulin-like growth factor (IGF) system in relaxin-induced growth of ovarian and uterine tissues. In the pig ovary, concentrations of relaxin that promote both theca and granulosa cell (GC) DNA synthesis in vitro also significantly (P < 0.05) increased GC IGF-I secretion. When IGF-I activity was blocked in the presence of an IGF-I antibody, the trophic effects of relaxin on GC [3H]thymidine incorporation into DNA were inhibited. However, there was no effect of relaxin on GC IGF binding proteins or IGF-I receptor. In the uterus, in vivo relaxin administration to prepubertal pigs resulted in the stimulation of growth and increases in uterine luminal IGF-I, IGF-II, and IGF binding proteins-2 and -3 secretion (P < 0.05). Thus, the trophic effects of relaxin on ovarian granulosa cells and the uterus involve tissue-specific changes in the IGF system. Additional studies are necessary to better understand the contribution of relaxin to follicular growth and uterine accommodation. These include characterization of the relaxin receptor and post-receptor binding events, as well as the potential impact of relaxin on other growth factor systems and how these systems interact to ultimately drive reproductive tissue growth.

Animals↗
PubMed1999

Relaxin secretion and gene expression in porcine granulosa and theca cells are stimulated during in vitro luteinization.

During formation of the corpus luteum, the primary source of relaxin switches from theca cells (TC) to granulosa-derived, large luteal cells. What controls this shift in relaxin production is poorly understood. The objective of this study was to observe the effect of luteinization on relaxin gene expression and secretion by porcine granulosa (GC) and TC using an in vitro model. TC and GC from medium-sized porcine follicles (4-6 mm) were treated for up to 8 days with LH (250 ng/ml) and/or insulin-like growth factor-I (IGF-I; 10 ng/ml). Media were assayed for relaxin and progesterone by RIA, changes in cell morphology were recorded, and total RNA was subjected to reverse transciption-polymerase chain reaction to monitor relaxin gene expression. In vitro luteinization, induced with LH + IGF-I treatment, was confirmed in both GC and TC by a change in morphology and a sustained, significant rise in progesterone secretion. In luteinizing GC, relaxin secretion was first detected after 5 treatment days, and steadily rose until it became significantly higher (p < 0.001) by treatment Days 7-8. In contrast, relaxin release from luteinizing TC was significant after only 2 days of treatment (p < 0.05) and increased consistently over the 8-day culture period (p < 0.001). In GC, relaxin mRNA was not detected until treatment Day 4 and became significantly higher (p < 0.001) by Day 8, the final treatment day. Relaxin transcript in luteinizing TC was low on treatment Days 2-4 and significantly higher (p < 0.01) by treatment Days 6 and 8. In summary, the present study demonstrates that hormones important in the control of luteinization are essential for regulating relaxin gene expression and secretion by GC and TC in the porcine follicle.

Animals↗
PubMed1999

Expression of connexin-26, -32, and -43 gap junction proteins in the porcine cervix and uterus during pregnancy and relaxin-induced growth.

Connexin (CX) proteins participate in growth, differentiation, and tissue remodeling. Relaxin-stimulated reproductive tissue growth and remodeling may be facilitated by enhanced intracellular communication. This study was an examination of the effects of relaxin in vivo on expression of CX-26, CX-32, and CX-43 in the cervix and uterus of prepubertal pigs. In addition, expression of these proteins was monitored in the sow uterus during pregnancy. Relaxin was administered to prepubertal gilts every 6 h for 54 h. CX expression was characterized by immunoblotting and localized by immunofluorescence. Significant increases in all three CXs were observed in the cervix following relaxin treatment (P < 0.05). Uterine CX proteins were also significantly higher (P < 0.05) in relaxin-treated animals compared to controls. The CX protein level in relaxin-treated animals was similar to that observed during the second half of pregnancy, but below levels found in mature, nonpregnant sows. This is the first evidence for specific CX expression in the porcine cervix, and the first study to show that relaxin increases the expression of CX proteins in the porcine uterus and cervix. The data show that CX proteins are differentially regulated in the uterus of the pig during pregnancy. These data support a role for CX-mediated communication during relaxin-induced reproductive tissue growth and remodeling.

Animals↗
PubMed1998

Connexin 43 gap junction protein expression during follicular development in the porcine ovary.

Connexin 43, a member of the highly conserved connexin family of gap junction proteins, is expressed in the pig ovary. In other species, ovarian connexin 43 expression and phosphorylation are hormonally regulated. We characterized connexin 43 expression and phosphorylation in the ovaries of mature pigs during the estrous cycle and in prepubertal gilts during follicular development induced by eCG (750 IU)/hCG (500 IU; 72 h later). Ovarian connexin 43 protein expression and phosphorylation were examined by immunoblot analysis. Connexin 43 was localized to specific follicular cell types during development by immunofluorescence. While no change in total connexin 43 protein expression was seen during the cycle, connexin 43 phosphorylation was significantly higher (p < 0.05) during the late follicular stage of the cycle than during the early luteal and early to mid-follicular stages. In ovaries of eCG/hCG-primed prepubertal pigs, connexin 43 protein levels remained steady, while phosphorylation of the protein increased significantly at 72 h and 84 h after eCG treatment (p < 0.05), then declined to pretreatment levels by 96 h (24 h post-hCG administration). Immunoreactive connexin 43 was localized predominantly to granulosa cells of cyclic pigs and eCG/hCG-primed prepubertal gilts. Follicular connexin 43 was highest between 60 h and 84 h after eCG and declined after hCG administration. Connexin 43 was not detected in morphologically atretic follicles, stroma, or vascular tissue of the ovary. This is the first evidence that porcine ovarian connexin 43 phosphorylation is differentially regulated during follicular development. The results suggest that hormonally induced changes in connexin 43 phosphorylation may play a coordinating role in porcine follicular development.

Animals↗
PubMed1998

Regulation of urokinase- and tissue-type plasminogen activator by relaxin in the uterus and cervix of the prepubertal gilt.

Changes in plasminogen activator are associated with the reproductive tissue remodelling that occurs during growth. Given the trophic effects of relaxin on the pig uterus and cervix, the present study was designed to examine the impact of relaxin on urokinase and tissue-type plasminogen activator (uPA and tPA) protein and activity in the uterus and cervix of prepubertal pigs. After relaxin administration in vivo to induce growth of the immature uterus and cervix, plasminogen activator activity was measured in uterine flushes and uterine and cervical tissue using a chromogenic substrate assay. Immunoreactive uPA and tPA protein in uterine flushes and uterine and cervical tissue was detected by western blotting. Urokinase plasminogen activator activity was significantly higher (P < 0.05) in uterine flushes from relaxin-treated animals than in controls. However, there was no change in uterine flush tPA activity or protein in response to in vivo relaxin treatment. There was no evidence for acid-labile inhibitors of plasminogen activator in uterine flushes of any of the animals. Cell-associated uterine tissue uPA and tPA activity, as well as protein, were similar in relaxin-treated and control prepubertal pigs. In the cervix, cell-associated tPA activity decreased significantly (P < 0.05) in relaxin-treated animals, while cervical uPA activity was unchanged. These results support the view that at least one means by which relaxin promotes pig uterine growth is by increasing uterine secretion of uPA. In addition, these studies suggest that relaxin administration in vivo to prepubertal gilts has tissue-specific effects with respect to plasminogen activator.

Animals↗
PubMed1998

Relaxin protein and gene expression in ovarian follicles of immature pigs.

Relaxin production by the ovarian follicle of gonadotropin-primed, prepubertal gilts is well documented. As far as we are aware, a source of relaxin in pig follicles, independent of gonadotropins, has not yet been reported. Therefore, the objective of this study was to determine whether relaxin is produced in porcine follicles in the absence of exogenous or cyclic gonadotropins. In immature pigs, immunoreactive relaxin was detected in fluids from small (1-3 mm), medium (4-5 mm) and large (>6 mm) follicles and localized to the theca interna of large follicles. Relaxin levels in follicular fluid significantly increased with follicle size (P<0.05). Relaxin mRNA was detected in whole small- and medium-sized follicles. In large follicles, the relaxin gene was expressed in thecal layers, but not granulosa cells. The abundance of relaxin transcript did not change with follicle size. In summary, relaxin protein and mRNA were detected in porcine follicles from immature animals, indicating that relaxin is produced in the porcine follicle in the absence of exogenous or cyclic gonadotropins. Relaxin's in vitro growth effects on porcine granulosa and theca cells support this follicular relaxin as a growth modulator during porcine follicular development.

Animals↗
PubMed1997

Relaxin increases insulin-like growth factors (IGFs) and IGF-binding proteins of the pig uterus in vivo.

Relaxin promotes growth of reproductive tissues, including the uterus. Although we have evidence of a role for insulin-like growth factor I (IGF-I) in mediating relaxin-induced growth of porcine granulosa cells in vitro, the mechanism of action by which relaxin enhances uterine growth has not been identified. To investigate a role for the uterine insulin-like growth factor (IGF) system in relaxin-induced uterine growth, we monitored the effects of relaxin on porcine IGFs and IGF-binding proteins (IGFBPs) in vivo. The trophic effects of relaxin on the uterus were elicited by administering relaxin or saline to prepubertal gilts every 6 h for 54 h. Three hours after the last injection, uterine flushes, uteri, follicular fluid, and ovaries were collected. Estradiol was measured in plasma and follicular fluid to confirm the prepubertal status of each animal. Significantly higher concentrations of uterine lumen IGF-I (P < 0.05) and IGF-II (P < 0.01) were observed in animals treated with relaxin. However, relaxin administration did not affect uterine IGF-I and -II gene expression, as determined by a ribonuclease protection assay and Northern analysis, respectively. In uterine flushes, relaxin treatment increased an IGFBP doublet (33 and 34.5 kDa) and IGFBP-3. The uterine IGFBP doublet was identified as IGFBP-2 by immunoprecipitation. Plasma or follicular fluid IGFs and IGFBPs were unaffected by relaxin administration. In addition, relaxin did not influence IGF-I binding to its uterine receptor. This is the first study to demonstrate regulation of the pig uterine IGF system by relaxin. In conclusion, the data point to IGF-I, IGF-II, IGFBP-2, and IGFBP-3 as putative mediators of relaxin-induced uterine growth in the pig.

Animals↗
PubMed1996

Expression of epithelial cadherin in the developing and adult pig ovary.

Epithelial cadherin (E-cadherin) is one member of a family of intracellular calcium (Ca(2+))-dependent adhesion molecules that mediates selective cell-cell adhesion in a variety of species. Since changes in adhesive function accompany ovarian tissue development and remodeling, we were interested in studying the expression of E-cadherin during ovarian maturation in the pig. The objectives of this study were 1) to investigate the pattern of E-cadherin mRNA and protein expression during ovarian ontogeny in the pig, 2) to identify specific cells expressing E-cadherin in the mature porcine ovary, and 3) to compare E-cadherin expression in cells of morphologically healthy and atretic follicles. The results showed the presence of a 120-kDa protein, corresponding to E-cadherin, which was highest in fetal and neonatal ovaries and declined markedly (3- to 8-fold, p < 0.05) with maturity (16 wk to adult). In the adult ovary, E-cadherin was highest in ovarian surface epithelium (OSE) whereas in healthy follicles, granulosa cells had the highest levels. A significant decline (p < 0.05) in E-cadherin expression was evident in granulosa and theca cells from atretic follicles when compared with E-cadherin expression in cells of healthy follicles. A 4.2-kb E-cadherin transcript was detected in ovaries from 15-day-old pigs, and expression was markedly reduced (p < 0.05) by 16 wk of age. In the ovary of pregnancy, there was a faint E-cadherin mRNA signal, whereas in follicles the signal was stronger and uniform across follicle size. This is the first report that E-cadherin is expressed by the porcine ovary during ontogeny and that follicular and OSE cells of the adult ovary express the protein. Although the role of E-cadherin in the porcine ovary is unknown, the decline in E-cadherin expression in atretic follicular cells suggests that E-cadherin has a role in maintaining the structural integrity of the ovarian follicle during growth and development.

Aging↗
PubMed1995

Relaxin-induced deoxyribonucleic acid synthesis in porcine granulosa cells is mediated by insulin-like growth factor-I.

Relaxin stimulates in vitro DNA synthesis and cell proliferation of porcine granulosa cells (GC) and theca cells. The objective of the study reported here was to determine whether components of the ovarian insulin-like growth factor (IGF) system mediate relaxin's growth-promoting effects on porcine GC in vitro. In small follicle GC, relaxin (1-100 ng/ml) significantly (p < 0.05) increased IGF-I secretion to 25-34% above control. Hormonal responsiveness of GC was shown by incubation with FSH (200 ng/ml), which resulted in 125% stimulation of IGF-I secretion relative to that in cells incubated alone. When IGF-I activity in the GC cultures was neutralized with a specific IGF-I antibody, relaxin (10 and 100 ng/ml)-induced [3H]thymidine incorporation was inhibited (p < 0.05). Coincubation with IGF-I antibody also suppressed basal and IGF-I (10 ng/ml)-induced [3H]thymidine incorporation into GC DNA, but had no effect on insulin (1 microgram/ml)-induced DNA synthesis, demonstrating the specificity and lack of toxicity of the IGF-I antibody. Ligand blot analysis showed no change in secretion of GC IGF binding protein (IGFBP) in response to relaxin (1, 10, and 100 ng/ml). In contrast, IGF-I (10 ng/ml) increased secretion of IGFBP-3 and -5, whereas FSH (200 ng/ml) decreased IGFBP-3 secretion and increased IGFBP-4 secretion (p < 0.05). In IGF-I receptor competition studies, IGF-I, but not relaxin, displaced [125I]IGF-I from the GC IGF-I receptor. These studies provide direct evidence for an interaction of relaxin and the ovarian IGF system. They are the first to show 1) a stimulatory effect of relaxin on IGF-I secretion; 2) the necessity of IGF-I activity for relaxin-induced GC DNA synthesis; and 3) the absence of an effect of relaxin on GC IGFBPs or IGF-I receptor. These findings support a paracrine role for relaxin in the porcine follicle and show that relaxin acts indirectly to promote follicle growth by stimulating GC IGF-I secretion.

Animals↗
PubMed1993

Relaxin stimulation of porcine granulosa cell deoxyribonucleic acid synthesis in vitro: interactions with insulin and insulin-like growth factor I.

Relaxin (RLX) is a product of the theca interna layer of the developing pig follicle. However, its physiological role within the ovary is unknown. The purpose of this study was to determine the effects of RLX alone or in conjunction with insulin and insulin-like growth factor-I (IGF-I) on porcine granulosa cell (GC) DNA synthesis and proliferation in vitro. The results showed RLX (1-1000 ng/ml), insulin (1-1000 ng/ml), and IGF-I (1-30 ng/ml) alone stimulated [3H]thymidine incorporation into DNA by GC from small, medium, and large porcine follicles (P < 0.05). The effect of RLX in combination with insulin or with IGF-I on DNA synthesis by GC from small and medium follicles was greater than the effect of either single factor (P < 0.05). In the large follicle, RLX increased GC DNA synthesis synergistically with insulin and IGF-I. FSH (100 ng/ml) or estradiol (10(-7) M) had no effect on basal or RLX-induced [3H]thymidine incorporation into GC DNA at any stage of follicular development. After 6 days incubation in vitro, RLX dose-dependently increased proliferation of GC from small follicles (P < 0.05). The increased cell number in response to RLX correlates with RLX-induced [3H]thymidine incorporation into DNA by these GC. Hydroxyurea, an inhibitor of DNA synthesis, blocked RLX-induced thymidine incorporation, indicating that the increase in thymidine incorporation in response to RLX was due to DNA synthesis and not repair. This is the first demonstration that RLX acts as a growth factor in the porcine follicle by increasing GC DNA synthesis and proliferation in vitro. RLX enhanced the growth-promoting effects of insulin and IGF-I on GC, which suggests an interaction of RLX with these proteins during follicular growth. These studies support the concept that locally produced thecal RLX alone or in combination with other growth effectors may play a key role in GC growth during porcine follicular development.

Animals↗
PubMed1993

Developmental expression of the relaxin gene in the porcine corpus luteum.

Northern analysis and in-situ hybridization were used to follow the development of relaxin gene expression in the newly forming corpus luteum (CL) after ovulation and throughout luteal development. Alkaline phosphatase (AP) was used as a marker of theca-derived lutein cells and the relationship between AP-positive and relaxin mRNA-containing cells was assessed. Ovaries from prepubertal pigs treated with pregnant mares serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG) were collected during the periovulatory period and at various times during 19 days after ovulation. In addition, CL from cyclic pigs on days 10 and 16 were used to monitor relaxin gene expression in small and large luteal cells. Northern analysis revealed that relaxin gene expression increased with CL development in the PMSG/hCG-treated pig, reaching maximal levels at around day 14 post-ovulation. Thereafter, as the CL regressed, the level of relaxin mRNA declined. In CL from cyclic pigs at day 10 of the cycle, only small luteal cells expressed relaxin mRNA. However, by day 16 of the cycle, large luteal cells were the source of relaxin gene expression. In-situ hybridization studies revealed that in the early CL (up to 30 h post-ovulation), the relaxin gene transcript was observed in cells along the margins of the CL and in the core of the infolding follicle wall corresponding to the AP-positive, luteinized theca cell layer. As luteinization progressed, the theca and granulosa cell layers could no longer be distinguished morphologically (from 54 h after ovulation until day 9). However, the pattern of relaxin hybridization persisted along the periphery in bands of cells penetrating the CL, and coincided with areas of AP staining, indicating that the theca lutein cells were the site of relaxin gene expression. At day 14, relaxin hybridization and AP staining were distributed throughout the luteal tissue. With CL regression both AP staining and relaxin hybridization declined. This pattern of relaxin hybridization in the CL of the gonadotrophin-primed pig was identical to that observed in cyclic pigs on days 10 and 16 of the cycle. These findings indicate that theca interna cells retain their ability to express the relaxin gene following ovulation and luteinization. In the early CL, the small theca-derived lutein cells are the source of relaxin transcript. However, as the CL becomes fully differentiated, the large granulosa-derived lutein cells acquire the capacity to express the relaxin message.

Alkaline Phosphatase↗
PubMed1993

Sources and biological actions of relaxin in pigs.

Although the major source of relaxin in pigs is the corpus luteum of pregnancy, there is now evidence for relaxin gene expression and translation into protein in the theca interna cells of the preovulatory follicle, the corpus luteum of the cycle and the uterus. The theca interna cells retain their ability to express the relaxin gene and protein following ovulation. During the early stages of development of the corpus luteum, the theca-derived small lutein cells are the source of the relaxin transcript. As the corpus luteum becomes fully functional, there is a switch in the site of relaxin synthesis from small theca-derived lutein cells to large granulosa-derived cells. In the absence of luteolysis, this switch is accompanied by a dramatic rise in relaxin synthesis. Relaxin has been identified in boar seminal plasma and can maintain or increase sperm motility. However, a source of relaxin in the boar has not been identified. Relaxin is an important regulator of uterine function during pregnancy acting systemically to suppress myometrial activity and promote cervical dilation at parturition. The changes in thecal relaxin production during follicle development and its ability to promote growth and changes in proteolytic enzyme activity of granulosa cells in vitro have led to the concept of an autocrine or paracrine role for relaxin within the follicle. Uterotrophic effects of relaxin have been reported in rodents and swine and support the hypothesis that relaxin promotes uterine growth and expansion in early pregnancy to accommodate the growing fetuses. Mammotrophic effects of relaxin in rodents have now been extended to pigs, with evidence that relaxin is necessary for normal mammary parenchymal development in late pregnancy. In most instances the mechanisms responsible for, and the physiological significance of, these diverse biological effects remain to be elucidated.

Animals↗