Secretion of angiogenic activity and progesterone by ovine luteal cell types in vitro

In the first experiment, minced luteal tissues from cyclic ewes (n = 5) were incubated for 6 h. Media conditioned by these luteal tissue explants stimulated proliferation and migration of endothelial cells. In a second experiment, corpora lutea (CL) from superovulated ewes (n = 12) were dissociated (two ewes/dispersion) and separated into three fractions: a non-elutriated fraction containing a mixed population of luteal cells, a fraction enriched with small steroidogenic luteal cells, and a fraction containing primarily large steroidogenic luteal cells. Fractions (2 X 10(5) viable steroidogenic luteal cells per milliliter of medium) were incubated with LH in doses of 0, .1, 1, 10, and 100 ng/ml for 7 d. Conditioned media were collected on d 1, 3, 5, and 7 of incubation. Across all days of incubation, media from small luteal cells stimulated proliferation of endothelial cells. Media from large luteal cell incubations, however, secreted an endothelial mitogen only on d 7 of culture. Mixed luteal cell cultures secreted mitogenic activity on d 3, 5, and 7 of incubation, but not on d 1. Luteinizing hormone did not influence release of mitogenic activity by any luteal cell fraction. Across all days of incubation, media from large luteal cells contained more progesterone than those from small luteal cells (528 +/- 137 vs 48 +/- 16 ng/ml with no LH). Mixed (non-elutriated) and small luteal cells increased progesterone secretion in response to LH, and this response was maintained during long-term culture. Large luteal cells did not increase progesterone secretion in response to LH. Steroidogenic activity of all cell types decreased as incubation time progressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.     

Growth hormone, insulin and glucose concentrations in bovine fetal and maternal plasmas at several stages of gestation

For cows on d 137 (n = 6), 180 (n = 8), 226 (n = 9) and 250 (n = 5) of gestation (Exp. 1), concentrations of insulin and glucose were two- to three-fold less (P less than .01) in fetal venous plasma than in uterine arterial plasma. Concentrations of growth hormone, conversely, were 10- to 20-fold greater (P less than .01) in fetal venous than in uterine arterial plasma. Concentrations of insulin and glucose in maternal and fetal plasmas and concentrations of growth hormone in maternal plasma did not vary with stage of gestation. Concentrations of growth hormone in fetal venous plasma, however, were greater on d 226 and 250 than on d 137 and 180. For cows (n = 6) on d 198 of gestation (Exp. 2), concentrations of insulin and glucose in maternal and fetal plasmas and of growth hormone in maternal plasma remained relatively constant in samples collected every 30 min for 3 h. In contrast, growth hormone concentrations in fetal venous plasma were highly variable and appeared to be episodic, with pulses of 10 to 60 ng/ml in amplitude. No significant correlations were found among concentrations of insulin, glucose and growth hormone in fetal venous plasma. When samples were collected every 15 min for 4 h from cows (n = 5) on d 198 of gestation (Exp. 3), episodes of growth hormone in fetal venous plasma were irregular in amplitude and frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumour Necrosis factor-α Receptors are Present in the Corpus Luteum Throughout the Oestrous Cycle and During the Early Gestation Period in Pigs

To determine the physiological significance of tumour necrosis factor‐α (TNFα) in the regulation of luteal functions in pig, this study was conducted to identify the presence of functional TNFα receptors in porcine corpora lutea (CL) throughout the oestrous cycle and the early gestation. The CL were isolated from pigs on days 4, 6, 8, 12 or 15 of the oestrous cycle (n=3; day 0 = oestrus) and days 15, 20 or 25 of gestation (n=3; day 0 = mating). A Scatchard analysis revealed the presence of a high‐affinity binding site for TNFα in all samples (dissociation constant; 2.7 ± 0.51 to 5.8 ± 0.50 nM ). The concentration of TNFα receptors was higher on day 15 of the oestrous cycle than on days 4 and 8 of the oestrous cycle (p < 0.05). Furthermore, TNFα receptor concentrations in the CL on days 15, 20 and 25 of gestation were significantly lower than on day 15 of the oestrous cycle (p < 0.05). On day 9 of the oestrous cycle, exposure of cultured luteal cells to 0.06–60 nM TNFα stimulated prostaglandin (PG) F_2α and PGE₂ secretion in a dose‐dependent manner (p < 0.05). These results indicate that functional TNFα receptors are present in the porcine CL throughout the oestrous cycle and early gestation, and suggest that TNFα plays one or more physiological roles in regulating CL function throughout the oestrous cycle and the early gestation period. In addition, TNFα receptor concentration in the CL of the late luteal stage (day 15) of the oestrous cycle was higher than on the respective day in the early pregnant pig, suggesting that TNFα plays a role in accomplishing luteolysis in the porcine CL.     

Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum

Angiogenesis in the developing corpus luteum (CL) is a prerequisite for establishment and maintenance of an early pregnancy. To explore the physiological significance of insulin-like growth factor-binding protein-7 (IGFBP7) in the developing CL, the effects of IGFBP7 on vascular endothelial growth factor (VEGFA)- and luteinizing hormone (LH)-induced in vitro tube formation were tested using isolated luteal microvascular endothelial cells (LECs). Capillary-like tube formation of LECs and their proliferation were stimulated by both VEGFA and LH. IGFBP7 treatment suppressed VEGFA- or LH-induced tube formation. The proliferation and migration of LECs, and phosphorylation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase 1/2 were inhibited by IGFBP7. Furthermore, IGFBP7 attenuated VEGFA-enhanced cyclooxygenase (COX)-2 mRNA expression and prostaglandin E₂ secretion. These findings suggest the possibility that luteal IGFBP7 secretion may suppress the stimulatory effect of VEGFA on angiogenesis in the early CL.     

Luteinizing hormone and progesterone concentrations and induction of estrus after use of norgestomet ear implants or constant infusion of gonadotropin-releasing hormone in anestrous, nonlactating dairy goats

Plasma luteinizing hormone and progesterone concentrations, time to onset of estrus, and pregnancy rates were determined in nonlactating anestrous does given 1 of 4 treatments: subcutaneous ear implants containing 3 mg of norgestomet for 9 days (NOR; n = 6); subcutaneous administration, using osmotic minipumps, of 250 ng of gonadotropin-releasing hormone (GnRH)/h for 48 hours (GnRH; n = 6); 3 mg of NOR for 9 days, followed immediately by 250 ng of GnRH/h for 48 hours (NOR + GnRH; n = 6); or no treatment (control; n = 6). During the 72-hour period after removal of NOR or insertion of GnRH pumps, 6 of 6, 0 of 6, 6 of 6, and 3 of 6 does were observed in estrus at a mean (+/- 13.8) hours in groups NOR, GnRH, NOR + GnRH, and control, respectively. Time from end of treatment to peak concentrations of luteinizing hormone were 56 +/- 4.0, 28 +/- 4.7, 34 +/- 4.3, and 41 +/- 9.7 hours (mean +/- SE) for NOR, GnRH, NOR +/- GnRH, and control, respectively. Peak concentrations of luteinizing hormone were significantly greater and occurred significantly later in does given NOR. Progesterone concentrations in does that became pregnant increased to concentrations greater than or equal to 1.0 ng/ml 3 to 5 days after breeding and remained high. Functional corpora lutea (CL) was found in 6 does that did not become pregnant, 1 CL was associated with pseudopregnancy and 1 CL was associated with ovulation prior to placement of the GnRH pumps. Functional CL failed to form in 10 of the 12 doses in groups GnRH and control.(ABSTRACT TRUNCATED AT 250 WORDS)     

The yin and yang of corpus luteum-derived endothelial cells: balancing life and death

A dense network of capillaries irrigates the corpus luteum (CL) allowing an intricate cross talk between luteal steroiodgenic and endothelial cell (EC) types. Indeed, luteal endothelial cells (LEC) play pivotal roles throughout the entire CL life-span. Microvascular endothelial cells are locally specialized to accommodate the needs of individual tissues, therefore unraveling the characteristics of LEC is imperative in CL physiology. Numerous studies demonstrated that endothelium-derived endothelin-1 (ET-1) is upregulated by the luteolytic hormone-prostaglandin F2alpha (PGF2alpha) and functions as an important element of the luteolytic cascade. To have a better insight on its synthesis and action, members of ET system (ET-1, ET converting enzyme -ECE-1 and ET(A) and ET(B) receptors) were quantified in LEC. The characteristic phenotype of these cells, identified by high ET-1 receptor expression (both ET(A), ET(B)) and low ET-1 and ECE-1 levels, was gradually lost during culture suggesting that luteal microenvironment sustains the selective phenotype of its resident endothelial cells. Proper vascularization and endothelial cell activity per se are essential for normal CL function. Therefore, factors affecting vascular growth are expected to play major role in the regulation of luteal function. Concomitantly with the angiogenic process, luteal PGF2alpha and its receptors (PGFR) are induced and maintained during most of the CL life-span, suggesting a possible role of PGF2alpha in LEC proliferation and function. Dispersed LEC expressed PGFR and incubation with the prostaglandin stimulated mitogen-activated protein kinase (MAPK) signaling cascade. PGF2alpha activated p42/44 MAPK phosphorylation also in long-term cultured LEC. In this cell type, PGF2alpha increased cell number, 3H-Thymidine incorporation and cell survival. Additionally, PGF2alpha rapidly and transiently stimulated the expression of immediate-early response genes, i.e. c-fos and c-jun mRNA, further suggesting a mitogenic effect for this prostaglandin in LEC. These data imply that PGF2alpha may assume different and perhaps opposing roles depending on luteal microenvironment.     

Effects of maternal nutrition and stage of gestation on body weight, visceral organ mass, and indices of jejunal cellularity, proliferation, and vascularity in pregnant ewe lambs

Peripubertal ewe lambs (44.3 +/- 1.1 kg of initial BW) were used in a 2 x 3 factorial design to test the effects of plane of nutrition (diet) and stage of gestation on maternal visceral tissue mass, intestinal cellularity, crypt cell proliferation, and jejunal mucosal vascularity. Singleton pregnancies to a single sire were established by embryo transfer, and thereafter ewes were offered a control (Control) or high (High) amount of a complete diet (2.84 Mcal/kg and 15.9% CP; DM basis) to promote slow or rapid maternal growth rates. After d 90 of gestation, feed intake of the Control group was adjusted weekly to maintain BCS and meet the increasing nutrient demands of the gravid uterus. Ewes were slaughtered at 50 d (n = 6 Control; n = 5 High), 90 d (n = 8 Control; n = 6 High), or 130 d (n = 8 Control; n = 6 High) of gestation. Ewes were eviscerated and masses of individual organs were recorded. The jejunum was sampled and processed for subsequent analyses. Final ewe BW for Control-fed ewes was similar at d 50 and 90 and increased (P = 0.10) from d 90 to 130 (46.0, 48.9, and 58.2 +/- 1.6 kg, respectively), whereas final BW increased (P 相似文献   

Production of mitogenic factors by cell types of bovine large estrogen-active and estrogen-inactive follicles

This study investigated whether large follicles (estrogen-active and estrogen-inactive) of cows produce factors with mitogenic activity. Large, preovulatory follicles (greater than or equal to 9 mm in diameter) were classified as estrogen-active or -inactive based on ratio of estrogen: progesterone concentrations in follicular fluid. After incubation of granulosa cells and thecal tissues from follicles, granulosa cell conditioned media (GCM), thecal conditioned media (TCM) and follicular fluid (FFL) were evaluated for effects on proliferation of bovine aortic endothelial (BAE) and BALB/3T3 (3T3) cells. Pools of GCM, TCM and FFL stimulated proliferation of BAE and 3T3 in a dose-dependent fashion. Across all follicles (n = 20), GCM had greater stimulatory effect on proliferation of BAE than on proliferation of 3T3 (135 vs 115% of unconditioned media controls), whereas TCM stimulated proliferation of BAE and 3T3 to a similar extent (128 and 128%). Across type (GCM and TCM) of conditioned media, estrogen-active follicles stimulated proliferation of BAE more than proliferation of 3T3 (137 vs 121% of unconditioned media controls), whereas estrogen-inactive follicles stimulated proliferation of BAE and 3T3 to a similar extent (120 vs 122%). As observed for GCM, FFL across all follicles had a greater stimulatory effect on proliferation of BAE than on proliferation of 3T3 (159 vs 141%). Granulosa-conditioned media stimulated proliferation of BAE and 3T3 only when obtained from estrogen-active follicles; mitogenic activities of TCM and FFL were not influenced by type of follicle. These data demonstrate that granulosa cells of large preovulatory bovine follicles secrete a mitogenic factor(s) that is more stimulatory for proliferation of BAE than for 3T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between ultrasonographic image attributes, histomorphology and proliferating cell nuclear antigen expression of bovine antral follicles and corpora lutea ex situ.

The aim of this study was to examine the relationships between quantitative ultrasonographic image characteristics, histological attributes and cell proliferating ability of bovine antral follicles and corpora lutea (CL) ex situ. Bovine ovaries (n = 30) from animals at various reproductive states (metoestrus-early dioestrus, n = 8; mid-dioestrus, n = 12; oestrous phase of peripubertal heifers, n = 6; and pregnancy, n = 4) were collected at the slaughterhouse. High-resolution ultrasonographic images of the ovaries were obtained in the water bath, digitized and subjected to computerized image analyses. The analyses utilized line and spot techniques designed to determine pixel values of the follicular wall (the largest follicles >2 mm in diameter in each ovary) and CL, respectively. Individual ovarian structures were dissected and processed for histology and immunohistochemical detection of proliferating cell nuclear antigen (PCNA). The mean follicular diameter was negatively correlated with total cell density (r = -0.45, p < 0.05), granulosa layer thickness (r = -0.67, p < 0.001) and the percentage of PCNA-positive cells (r = -0.57, p < 0.001). Numerical pixel values and heterogeneity of the follicular wall were positively correlated with total cell density (r = 0.42, p < 0.05 and r = 0.62, p < 0.05; respectively), granulosa layer thickness (both r = 0.39, p < 0.05), and the percentage of PCNA-positive cells (r = 0.54, p < 0.01 and r = 0.69, p < 0.001, respectively). Estimates of cell density and proliferating cell index were not correlated with the ultrasonographic image attributes of CL. We conclude that follicular size and echotextural variables, as determined by computer-assisted image analysis of ovaries ex situ, are reliable markers of the histophysiological properties of bovine antral follicles, but the ultrasonographic characteristics are not indicative of cell density and proliferation in the bovine CL.     

Endocrine and cellular characteristics of corpora lutea from cows with a delayed post-ovulatory progesterone rise

The timing of the post-ovulatory progesterone rise is critical to the embryonic development and survival. The aim of this study was to determine the underlying causes of delayed post-ovulatory progesterone rises. Two groups of non-lactating dairy cows with early (n = 11) or late (n = 9) post-ovulatory progesterone rises were created by inducing luteolysis in the presence of either a large (> 10 mm) or small (< 10 mm) follicle, respectively. LH pulses were measured on days 4 (all cows) and 7 (n = 7, early; n = 5, late) (day 1= ovulation). The cows were slaughtered on day 5 (n = 4 each group) or 8 (n = 7, early; n = 5, late). Immunohistochemical analysis for endothelial cells (von Willebrand Factor, VWF), steroidogenic cells (3beta-HSD) and proliferation marker (Ki67) were performed. The basal progesterone production and LH responsiveness (0.001-100 ng/ml) of dispersed luteal cells was investigated. The luteal concentrations of FGF-2 and VEGF were measured by ELISA and RIA, respectively. There were no differences in LH pulse characteristics, area of VWF staining, proliferation index, steroidogenic cell characteristics, basal or LH-stimulated progesterone production by luteal cells between cows with an early or late progesterone rise (P > 0.10). However, the area of VWF staining increased from days 5 to 8, while the proliferation index decreased (P < 0.05). Furthermore, the luteal cells were more responsive to LH on day 8 (P < 0.01). Luteal concentrations of FGF-2 were higher on day 5 (P = 0.05), while VEGF was greater on day 8 (P < 0.01). In conclusion, we have clearly shown that LH support, degree of vascularization or luteal cell steroidogenic capacity were not the major factors responsible for inadequate secretion of progesterone by the developing bovine CL.     

Ovarian function in ruminants

The purpose of this overview is to highlight important steps of ovarian regulation during follicle development, ovulation and the life span of corpus luteum (CL) in ruminants. The ovarian cycle is central to reproductive function. It is characterized by repeating patterns of cellular proliferation, differentiation and transformation that encompass follicular development and ovulation as well as the formation, function and regression of the CL. In the first part, the importance and regulation of final follicle growth and especially of angiogenesis and blood flow during folliculogenesis, dominant follicle development and CL formation are described. Our results underline the importance of growth factors especially of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) for development and completion of a dense network of capillaries (angiogenesis) during follicle growth and CL formation. In the second part, the regulation of CL function by endocrine/paracrine and autocrine acting regulators is discussed. There is evidence that besides the main endocrine hormones luteinizing hormone (LH) and growth hormone (GH) local regulators as growth factors, peptides, steroids and prostaglandins are important modulators of luteal function. During early CL development until midluteal stage oxytocin (OT), prostaglandins and progesterone (P) itself stimulate luteal cell proliferation and function supported by the luteotropic action of a number of growth factors. The still high mRNA expression, protein concentration and localization of VEGF, FGF and IGF family members in the cytoplasm of luteal cells during midluteal stage suggest that they play pivotal role in the maintenance (survival) of this endocrine tissue. The major function of the CL is to secrete P. Progesterone itself regulates the length of the estrous cycle via influencing the timing of the luteolytic PGF2alpha signal from the endometrium. At the end of a nonfertile cycle, the regression of CL commences, steroidogenic capacity is lost (functional luteolysis), cell death is initiated, and tissue involution as well as resorption occurs within a few days (structural luteolysis). The cascade of mediators during luteolysis is very complex and still awaits elucidation. Evidence is given for participation of blood flow, inflammatory cytokines, vasoactive peptides (angiotensin II and endothelin-1), and decrease of the classical luteotropic mediators.     

Ovulation of the preovulatory follicle originating from the first-wave dominant follicle leads to formation of an active corpus luteum

The objective of our study was to compare the characteristics of the corpus luteum (CL) formed after ovulation of the dominant follicle (DF) of the first follicular wave (W1) and those of the CL formed after ovulation of the DF of the second (induced) follicular wave (W2). Non-lactating Holstein cows were used for this study. In Experiment 1, cows were treated with PGF2α and GnRH on days 6 and 8 (day 0 = day of follicular wave emergence) for W1 (n = 6) and W2 (n = 6), respectively. Dominant follicles were aspirated on day 9 to quantify the amounts of mRNA (VEGF120, VEGF164, FGF-2, StAR, P450-scc and 3β-HSD) in granulosa cells (GC). In Experiment 2, the size and blood flow area of the CL formed after ovulation of the DF in W1 (W1CL; n = 6) and W2 (W2CL; n = 6) (the day of DF ovulation in W1 and W2 was day 10) were evaluated on days 12, 15, 18 and 21. The plasma P4 concentration was measured on days 10 to 21. The amounts of VEGF164, P450-scc and 3β-HSD mRNA were higher (P < 0.05) in the DF in W1, and those of VEGF120,FGF-2 and StAR mRNA tended to be higher (P < 0.1) in the DF in W1. The size of the CL was greater in the W1CL on days 15, 18 and 21. The blood flow area of the CL was greater in the W1CL on days 12 and 15. The plasma P4 concentrations were higher in the W1CL. These results indicate that the CL formed after ovulation of the DF in W1 was greater in terms of size, blood flow and plasma P4 concentration.     

Impacts on conceptus survival in a commercial swine herd

An estimated 30 to 40% of potential piglets are lost before farrowing in U.S. or European pig breeds. Because these studies were conducted in limited numbers of university research herds, we decided to characterize the timing, pattern, and extent of conceptus loss in a commercial swine herd in Iowa (Pig Improvement Company; Camborough Line). Sows (parities 2 to 14) were slaughtered on d 25 (n = 83), 36 (n = 78), or 44 (n = 83) of gestation. These days coincide with periods before, during, and after uterine capacity becomes limiting to conceptus survival. At slaughter, numbers of corpora lutea (CL) and uterine horn length were determined, and conceptuses were removed and evaluated. Uterine horn length and CL number did not differ among these days of gestation, averaging 217 cm and 26.6 CL, respectively. In contrast, numbers of conceptuses decreased (P < 0.05) from 15.8 on d 25 to 12.9 on d 36, then remained relatively constant through d 44 (12.1). Thus, conceptus survival averaged 60.2% on d 25, 50.1% on d 36, and 46.3% on d 44, based on numbers of CL present. There was a positive correlation (P < 0.001; r = 0.50) between numbers of viable conceptuses on d 25 and ovulation rate, but this association was completely lost by d 36 (P > 0.10) when uterine capacity becomes limiting. In agreement with this premise, uterine horn length and conceptus number were not associated on d 25 but exhibited positive correlations (P < 0.05) on d 36 (r = 0.36) and d 44 (r = 0.40). On all 3 d examined, the numbers of viable conceptuses were not associated with fetal weight but were negatively correlated (P < 0.05) with placental weight. Compared with the commonly reported values for ovulation rate and percentage conceptus loss in university research herds, values from these production animals were extremely high. Data suggest that throughout this period, larger litters were associated with conceptuses exhibiting small placentae. These data lend support to the concept that increased placental efficiency (fetal weight/placental weight) may contribute to increased litter size in the pig.     

Retention of a functional corpus luteum and peripheral concentrations of 13,14-dihydro-15-keto-prostaglandin F2alpha following metestrus administration of Syncro-Mate-B.

This study was conducted to examine the effects of metestrus administration of SyncroMate-B (SMB) on PGF2alpha secretion and corpus luteum (CL) development. In a study replicated over 2 yr, cows were observed for spontaneous estrus in yr 1, and cows received an injection of 25 mg of PGF2alpha and were observed for subsequent estrus in yr 2. At standing estrus (estrus = d 1), cows were randomly allotted to receive either the standard SMB regimen (n = 40) on d 3 of the estrous cycle or no treatment (n = 8). Fifty percent (n = 20) of SMB-treated cows were administered PGF2alpha on d 10 of the estrous cycle 48 h prior to implant removal. Twice-daily blood samples were collected in the morning (AM) and evening (PM) from d 2 AM through d 14 AM of the treated estrous cycle and subsequently analyzed for progesterone (P4) and PGF2alpha metabolite (PGFM). Prior to statistical analysis, SMB- and SMB/PGF2alpha-treated cows were sorted according to P4 concentration at d 10 of the treated estrous cycle to either a CL functional group (P4 > or = 1 ng/mL; n = 20) or a CL nonfunctional group (P4 < 1 ng/mL; n = 17). Following d 10 AM administration of PGF2alpha, functional and nonfunctional groups were further subdivided based on treatment. The groups were as follows: untreated control cows (n = 8); SMB-treated cows retaining a functional CL (SMB-F; n = 8); SMB-treated cows with a nonfunctional CL (SMB-N; n = 11); SMB/PGF2alpha-treated cows retaining a functional CL (SMB/PG-F; n = 12); and SMB/PGF2alpha-treated cows with a nonfunctional CL (SMB/PG-N; n = 6). Of all SMB-treated cows, 54% retained a functional CL through d 10 AM of the treated estrous cycle. Mean serum P4 concentrations increased for cows in all groups until d 7, after which P4 concentrations increased for cows in SMB/PG-F, SMB-F, and control groups and decreased for cows in SMB/PG-N and SMB-N groups. Following PGF2alpha administration on d 10, mean serum P4 concentrations remained < 1 ng/mL for cows in SMB/PG-N and SMB-N groups, decreased to < 1 ng/mL for cows in the SMB/ PG-F group, and remained > 1 ng/mL for cows in SMB-F and control groups. Mean serum PGFM concentrations tended (P = .06) to increase in cows with nonfunctional CL compared with control cows on d 8 AM and were greater (P < .05) in cows with functional CL on d 8 PM through d 9 PM. These results indicate that retention of a functional rather than a nonfunctional CL following metestrus administration of SMB is dependent on a premature release of uterine PGF2alpha.     

Changes in metabolic and reproductive characteristics associated with lactation and glucose infusion in the postpartum ewe

This study examined mechanisms whereby the metabolic environment interacts with basic reproductive function. Ewes lambing during the breeding season were fed to maintain (MAINT, n = 10) or gain (GAIN, n = 11) body weight during the last 4 mo of gestation. From d 7 to 22 postpartum, ewes were infused iv with saline (n = 10) or glucose at a rate calculated to increase normal glucose entry rate by 75% (n = 11). Blood samples were collected daily to determine plasma concentrations of nutritive metabolites and insulin and at frequent intervals on d 14 and 21 to determine serum gonadotropin concentrations. Hypothalami and pituitaries were collected on d 22 to determine hormone content and receptor concentrations. Plasma concentrations of nutritive metabolites and insulin indicated that MAINT ewes mobilized more (P less than .01) body fat and protein reserves during gestation and early lactation than ewes in the GAIN group. Glucose infusion elevated plasma concentrations of glucose (P less than .05) and insulin (P less than .07) and reduced (P less than .05) fat and protein mobilization, even though it depressed feed intake (P less than .001), compared with saline infusion. Hypothalamic gonadotropin-releasing hormone (GnRH), pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) content and pituitary GnRH receptor concentration were similar between treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Pregnancy and Foetal Number on Diameter of Corpus Luteum,Maternal Progesterone Concentration and Oxidant/Antioxidant Balance in Ewes

The aim of this study was to determine the changes in diameter of corpus luteum (CL), maternal progesterone (P) concentration, lipid peroxidation and non‐enzymatic antioxidant levels along with enzymatic antioxidant activities in pregnant ewes bearing single and twin foetuses. The ewes were selected from healthy animals that were brought to the abattoir for slaughtering. The ewes were divided into three groups: Group 1 (non‐pregnant, non‐oestrous, n = 30), Group 2 (pregnant bearing a single foetus, n = 30) and Group 3 (pregnant bearing twin foetuses, n = 12) after they were slaughtered. Pregnant ewes were in the first half of the pregnancy. The diameter of CL and P concentration of pregnant ewes bearing a single foetus or twin foetuses were found higher than that found in non‐pregnant ewes. Similarly, the P concentration of pregnant ewes bearing twin foetuses was higher than that found in pregnant ewes bearing a single foetus. Malondialdehyde (MDA) level in pregnant ewes bearing twin foetuses was higher than that found in both non‐pregnant and pregnant ewes bearing a single foetus. The serum glutathione (GSH) level and glutathione‐peroxidase (GSH‐Px) activity of pregnant ewes bearing twin foetuses were found lower than that found in non‐pregnant ewes. Additionally, the GSH‐Px activity of pregnant ewes bearing twin foetuses was found lower than that found in pregnant ewes bearing a single foetus. No significant difference was found between pregnant ewes bearing female and male foetus with respect to diameter of CL, P concentration and oxidative stress parameters. There were significant positive correlations between foetal number (0, 1, 2) and diameter of CL, P concentration, MDA level, and between P concentration and diameter of CL, MDA level. However, significant negative correlations were found between foetal number (0, 1, 2) and GSH level, GSH‐Px activity, and between P concentration and GSH‐Px activity. In conclusion, the diameter of CL enlarges, P production increases and oxidant/antioxidant balance impairs because of the gestation stress in ewes during pregnancy.     

Microvascularization and angiogenic activity of equine corpora lutea throughout the estrous cycle

Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE₂) and luteolytic (PGF₂) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the corpus albicans. Hyperplasia of luteal tissue increased until the mid luteal phase and it was followed by tissue regression. Luteal explants were cultured with no hormone added, or with PGF₂, LH, PGE₂, LH + PGE₂ or LH + PGF₂. Media conditioned by equine luteal tissue from different stages of the luteal phase were able to stimulate mitogenesis of bovine aortic endothelial cells (BAEC), suggesting the presence of angiogenic activity. No difference was observed among luteal structures on their mitogenic capacity, for any treatment used. Nevertheless, Late-CL conditioned-media with PGF₂ showed a significant decrease in BAEC proliferation (p < 0.05) and LH + PGF₂ a tendency to reduce mitogenesis. Thus, prostaglandin F₂ may play a role on vascular regression of the CL during the late luteal phase in the mare. These data suggest that luteal angiogenesis and vascular regression in the mare are coordinated with the development of non-vascular tissue and might be regulated by many different factors.     

Serum concentrations of pituitary and adrenal hormones in female pigs exposed to two photoperiods

Serum concentrations of pituitary and adrenal hormones were determined in lactating sows and ovariectomized (OVX) gilts exposed to 8 h (8L:16D) or 16 h of light (16L:8D). In addition serum prolactin (PRL) concentrations were determined after a thyrotropin releasing hormone (TRH) challenge. At 103 +/- 2 d of gestation or 3 wk after ovariectomy of nulliparous gilts on d 7 to 9 of the estrous cycle (d - 10), blood samples were collected from jugular vein cannulae at 30-min intervals for 8 h beginning at 0800 h. Immediately after the last sample, 13 sows and five OVX gilts were assigned to 8L:16D and 14 sows and five OVX gilts were assigned to 16L:8D/d and placed in two identical chambers in the farrowing house. Blood sampling was repeated on d 7, 14 and 21 of lactation in the sows and on d 7, 14, 21 and 28 in the OVX gilts. In Exp. 1, serum cortisol (C) concentrations were similar for sows exposed to 8L:16D (n = 7) and 16L:8D (n = 6) treatments, whereas in Exp. 2, serum C concentrations for sows exposed to 8L:16D (n = 6) were lower than those exposed to 16L:8D (n = 6) on d 7, 14 and 21. Photoperiod failed to influence serum concentrations of PRL, luteinizing hormone (LH) and growth hormone in the lactating sows or PRL in the OVX gilts. Photoperiod also failed to affect mean basal serum concentrations, peak height and peak frequency for PRL and LH in the lactating sows or for PRL in the OVX gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH up-regulates angiogenic factors in luteal cells of buffaloes

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4–6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.