Characterization of matrix metalloproteinase-2 and matrix metalloproteinase-9 and their inhibitors in equine granulosa cells in vivo and in vitro.

Matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) regulate tissue remodeling events necessary for ovulation. Thus, changes in MMP and TIMP expression and protein enzyme activity were examined in vivo and in vitro during follicular development and atresia in the horse. Equine granulosa cells and follicular fluid from medium (15 to 29 mm) healthy and atretic follicles and from large (>30 mm) healthy and preovulatory follicles were collected by transvaginal aspiration. The cells were either snap-frozen (in vivo study) or cultured for 48 h (in vitro study) to determine gene expression and protein enzyme activity of MMP-2 and MMP-9 and TIMP-1 and TIMP-2. Concentrations of progesterone and estradiol were determined by RIA in follicular fluid and conditioned media and were used along with follicle dynamics to classify follicles. In vivo, expression of MMP-2 and TIMP-2 was increased (P < 0.05) in large-preovulatory follicles, whereas TIMP-1 was decreased. The ratio of MMP-2:TIMP-2 expression was decreased (P < 0.05) in medium-healthy and large-preovulatory follicles, whereas the MMP-9:TIMP-1 ratio was increased only in large-preovulatory follicles compared with large-healthy follicles. Estradiol was greatest (P < 0.05) in the fluid of large-healthy and large-preovulatory follicles. However, medium-atretic follicles were associated with the least estradiol concentrations, both in vivo and in vitro. Progesterone concentrations were greatest (P < 0.05) in large-preovulatory follicles both in vivo and in vitro. In healthy follicles in vivo, the diameter was correlated with estradiol concentration, the estradiol:progesterone ratio, MMP-9 and TIMP-1 expression, and MMP-2 and MMP-9 protein activity. In contrast to in vivo studies, the ratio of MMP-9:TIMP-1 expression was increased (P < 0.05) in medium-healthy follicles; TIMP-2 expression decreased in large-preovulatory follicles in vitro. In addition, MMP-9 protein activity was decreased (P < 0.05) in the media samples of cells from large-healthy follicles compared with those from medium-healthy follicles. These results indicate that changes in MMP-2 and MMP-9 activities may be essential to the tissue reorganization necessary for ovulation in the equine ovary.

Relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse.

Recent studies associate obesity and insulin resistance in horses with development of abnormal reproductive function and debilitating laminitis. The factors contributing to insulin resistance in obese horses are unknown. However, human studies provide evidence that elevated inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha), IL1, and IL6 play direct roles in development of obesity-associated insulin resistance. Thus, inflammation may be a key link between obesity and insulin resistance in horses. The aim of the current investigation was to examine possible relationships between obesity, inflammatory cytokines, and insulin sensitivity (IS) in the horse. Age was recorded and BCS and percent body fat (% FAT) were determined as measures of obesity in 60 mares. In addition, blood mRNA expression of IL1, IL6, and TNFalpha and circulating concentrations of TNFalpha protein (TNFp) were determined in each mare. Finally, fasted concentrations of insulin were determined, and IS was determined using the hyperinsulinemic, euglycemic clamp. Significant correlations between several variables provided evidence for the design of 4 population regression models to estimate relationships between measures of obesity, inflammatory factors, and IS in the sample population. The results of these analyses revealed that IS decreased as BCS and % FAT increased (P < 0.001) in the sample population. Additionally, increased IL1 (P < 0.05) and TNFp (P < 0.01) were associated with decreased IS. However, increased TNFalpha (P < 0.001) was associated with decreased IS only in mares 20 yr of age and older. Increased BCS and % FAT were associated with increased expression of TNFalpha (P = 0.053) and IL1 (P < 0.05), and increased TNFp (P < 0.05). Surprisingly, increased BCS and % FAT were associated with decreased IL6 expression (P = 0.05) in mares <20 yr of age. Finally, evaluation of the influence of obesity and inflammatory cytokines on IS within the same model suggested that BCS and % FAT (P < 0.001) with TNFalpha [mRNA (P = 0.07) and protein (P < 0.05)] are inversely associated with IS independently of one another. Combined, these results provide the first evidence associating obesity with increased inflammatory factors in the horse. Furthermore, the results suggest that an interrelationship exists among obesity, inflammatory cytokines, and IS in the horse and emphasize the need for further studies to elucidate the nature of these relationships.

Obesity is associated with altered metabolic and reproductive activity in the mare: effects of metformin on insulin sensitivity and reproductive cyclicity.

In mares, obesity is associated with continuous reproductive activity during the non-breeding season. To investigate the effect of obesity and associated alterations in metabolic parameters on the oestrous cycle, two related studies were conducted. In Experiment 1, obese (body condition score > 7) mares were fed ad libitum or were moderately feed restricted during the late summer and autumn months. Feed restriction did not alter the proportion of mares entering seasonal anoestrus. However, obese mares exhibited a significantly longer duration of the oestrous cycle, significant increases in circulating concentrations of leptin and insulin, and decreased insulin sensitivity and concentrations of thyroxine compared with feed-restricted mares throughout the experiment. Experiment 2 was designed to investigate the effects of administration of the insulin-sensitising drug metformin hydrochloride on insulin sensitivity and the characteristics of the oestrous cycle in obese mares. In a dose-response trial, metformin increased insulin sensitivity after 30 days following administration of 3 g day(-1), but not 6 or 9 g day(-1), compared with controls receiving vehicle only. However, there were no differences in insulin sensitivity or oestrous cycle characteristics between control and metformin-treated groups when the 3 g day(-1) dose was tested for a longer period of 2 months. These results demonstrate that obesity is associated with aberrations in the oestrous cycle and perturbations in several markers of metabolic status. The results also indicate that metformin is not an effective long-term monotherapy for increasing insulin sensitivity in horses at the doses tested. Additional studies are needed to examine possible effects of increasing insulin sensitivity on reproductive activity in obese mares.

Development of a model for inducing transient insulin resistance in the mare: preliminary implications regarding the estrous cycle.

Peripheral insulin resistance is the failure of proper cellular glucose uptake in response to insulin. Insulin resistance and hyperinsulinemia are associated with several disease states in the horse and reproductive function disturbances in humans, including polycystic ovarian syndrome. To test the hypothesis that insulin resistance (IR) and hyperinsulinemia disrupt the estrous cycle in mares, two experiments were conducted to first develop a model to induce IR and to then examine the effect of this model on the duration of the estrous cycle. In Exp. 1, a hyperinsulinemic-euglycemic clamp (HEC) procedure was performed on seven mares to determine insulin sensitivity before and immediately following infusion of a heparinized lipid solution. The HEC procedure was repeated 1 wk after lipid infusion. Mares developed IR following the lipid infusion (P < 0.05), and some individuals maintained IR for up to 1 wk. Mares also exhibited increased blood insulin both immediately following treatment and 1 wk later (P < 0.05). In Exp. 2, induction of insulin resistance by lipid solution was not accompanied by changes in circulating concentrations of luteinizing hormone, and duration of the luteal phase, compared with the duration of untreated luteal phases. Nonetheless, lipid infusion and the resultant insulin resistance were associated with an increased interovulatory period (P < 0.05), and peak concentrations of progesterone (P < 0.05) were higher during the treated vs. untreated luteal phases of the estrous cycle. The results from the preliminary study suggest that infusion of a lipid solution may induce transient insulin resistance and hyperinsulinemia. The resulting insulin resistance and hyperinsulinemia may modify characteristics of the estrous cycle, perhaps at the level of the ovary.

Effect of short-term exercise training on insulin sensitivity in obese and lean mares.

Twelve untrained aged mares were used to determine whether 7 days of light exercise improved peripheral tissue insulin sensitivity. Mares were divided into obese-exercised (n = 3), obese-sedentary (n = 3), lean-exercised (n = 3) and lean-sedentary (n = 3) groups. The exercised groups were worked at a trot to a heart rate (HR) of not more than 140 beats/min for 30 min in a round pen. Each group was subjected to 3 euglycaemic hyperinsulinaemic clamps: prior to exercise (P), 24 h following the seventh exercise training session (E) and 9 days postexercise training (PE). Prior to exercise training, the mares in the obese group were confirmed insulin-resistant compared to the mares in the lean group. There was no change in bodyweight or body condition in the obese or lean groups throughout the study. Glucose infusion rate (GIR) was higher (P<0.05) on E compared to P days in the obese-exercised and lean-exercised groups. Insulin sensitivity returned to pre-exercise values by 9 days postexercise in the obese-exercised and lean-exercised groups. The results of this study suggest that improvement in insulin sensitivity occurs in obese mares without a long interval of exercise training and in the absence of a change in bodyweight.

Potential signals mediating the maintenance of reproductive activity during the non-breeding season of the mare.

The seasonal nature of reproductive activity in mares is widely accepted and considerable attention has focused on the mechanisms that lead to the initiation of the breeding season. In contrast, considerably less information is available about the termination of the breeding season. It is interesting to note that each winter a sub-population of mares continues to undergo oestrous cyclicity during the non-breeding season. Continuation of reproductive activity during the winter occurs most frequently in mares that maintain a non-pregnant condition in successive years. The maintenance of a non-pregnant condition in successive years leads to an increase in the percentage of total body fat and it has been proposed that the degree of adiposity may be a determinant of reproductive activity during the winter months. To investigate this hypothesis we have manipulated fat stores by either pharmacological treatments or feed restriction. The studies described in this review demonstrate that manipulation of body fat during the autumn months fails to modify the mechanisms that lead to anoestrus or the proportion of mares that continues to show oestrous cyclicity during the winter months. On the basis of these and related studies two hypotheses are presented that may serve as a template for future work. The first hypothesis proposes that one aspect of the long-term regulation of seasonal reproductive rhythms in mares, specifically anoestrus, may reflect recognition of the availability of metabolic fuels before perception of a change in photoperiod. Alternatively, energy availability may need to reach a critical value before a presumptive inhibitory daylength signal initiates termination of the breeding season. This review describes previous and current studies that have led to development of these proposals.