Assessment of luteal rescue and desensitization of macaque corpus luteum brought about by human chorionic gonadotrophin and deglycosylated human chorionic gonadotrophin treatment

The objective of the current study was to investigate the mechanism by which the corpus luteum (CL) of the monkey undergoes desensitization to luteinizing hormone following exposure to increasing concentration of human chorionic gonadotrophin (hCG) as it occurs in pregnancy. Female bonnet monkeys were injected (im) increasing doses of hCG or dghCG beginning from day 6 or 12 of the luteal phase for either 10 or 4 or 2 days. The day of oestrogen surge was considered as day ‘0’ of luteal phase. Luteal cells obtained from CL of these animals were incubated with hCG (2 and 200 pg/ml) or dbcAMP (2.5,25 and 100 M) for 3h at 37°C and progesterone secreted was estimated. Corpora lutea of normal cycling monkeys on day 10/16/22 of the luteal phase were used as controls. In addition thein vivo response to CG and deglycosylated hCG (dghCG) was assessed by determining serum steroid profiles following their administration. hCG (from 15–90 IU) but not dghCG (15-90 IU) treatment in vivo significantly (P < 0.05) elevated serum progesterone and oestradiol levels. Serum progesterone, however, could not be maintained at a elevated level by continuous treatment with hCG (from day 6–15), the progesterone level declining beyond day 13 of luteal phase. Administering low doses of hCG (15-90 IU/day) from day 6–9 or high doses (600 IU/day) on days 8 and 9 of the luteal phase resulted in significant increase (about 10-fold over corresponding control P < 0.005) in the ability of luteal cells to synthesize progesterone (incubated controls) in vitro. The luteal cells of the treated animals responded to dbcAMP (P < 0.05) but not to hCC added in vitro. The in vitro response of luteal cells to added hCG was inhibited by 0,50 and 100% if the animals were injected with low (15-90 IU) or medium (100 IU) between day 6–9 of luteal phase and high (600 IU on day 8 and 9 of luteal phase) doses of dghCG respectively; such treatment had no effect on responsivity of the cells to dbcAMP. The luteal cell responsiveness to dbcAMP in vitro was also blocked if hCG was administered for 10 days beginning day 6 of the luteal phase. Though short term hCG treatment during late luteal phase (from days 12—15) had no effect on luteal function, 10 day treatment beginning day 12 of luteal phase resulted in regain ofin vitro responsiveness to both hCG (P < 0.05) and dbcAMP (P < 0.05) suggesting that luteal rescue can occur even at this late stage. In conclusion, desensitization of the CL to hCG appears to be governed by the dose/period for which it is exposed to hCG/dghCG. That desensitization is due to receptor occupancy is brought out by the fact that (i) this can be achieved by giving a larger dose of hCG over a 2 day period instead of a lower dose of the hormone for a longer (4 to 10 days) period and (ii) the effect can largely be reproduced by using dghCG instead of hCG to block the receptor sites. It appears that to achieve desensitization to dbcAMP also it is necessary to expose the luteal cell to relatively high dose of hCG for more than 4 days     

A couple stress model of blood flow in the microcirculation

A simple mathematical model depicting blood flow in the capillary is developed with an emphasis on the permeability property of the blood vessel based on Starling's hypothesis. In this study the effect of inertia has been neglected in comparison with the viscosity on the basis of the smallness of the Reynolds number of the flow in the capillary. The capillary blood vessel is approximated by a circular cylindrical tube with a permeable wall. The blood is represented by a couple stress fluid. With such an ideal model the velocity and pressure fields are determined. It is shown that an increase in the couple stress parameter increases the resistance to the flow and thereby decreases the volume rate flow. A comparison of the results with those of the Newtonian case has also been made.     

Influence of electric field on the unsteady dispersion coefficient in couple-stress flow

The influence of electric field on unsteady convective diffusion in couple stress flow is studied using a time dependent dispersion model. The electric field, arising as a body couple in the governing equations, is shown to increase the axial dispersion coefficient. This is useful in the control of haemolysis caused by artificial organs implanted or extracorporeal. The contribution of pure convection in the dispersion of concentration is singled out and investigated in detail. The results obtained are compared with those in the absence of electric field and some important conclusions are drawn.     

Standardization and validation of an induced ovulation model system in buffalo cows: Characterization of gene expression changes in the periovulatory follicle

In bovines characterization of biochemical and molecular determinants of the dominant follicle before and during different time intervals after gonadotrophin surge requires precise identification of the dominant follicle from a follicular wave. The objectives of the present study were to standardize an experimental model in buffalo cows for accurately identifying the dominant follicle of the first wave of follicular growth and characterize changes in follicular fluid hormone concentrations as well as expression patterns of various genes associated with the process of ovulation. From the day of estrus (day 0), animals were subjected to blood sampling and ultrasonography for monitoring circulating progesterone levels and follicular growth. On day 7 of the cycle, animals were administered a PGF(2alpha) analogue (Tiaprost Trometamol, 750 microg i.m.) followed by an injection of hCG (2000 IU i.m.) 36 h later. Circulating progesterone levels progressively increased from day 1 of the cycle to 2.26+/-0.17 ng/ml on day 7 of the cycle, but declined significantly after PGF(2alpha) injection. A progressive increase in the size of the dominant follicle was observed by ultrasonography. The follicular fluid estradiol and progesterone concentrations in the dominant follicle were 600+/-16.7 and 38+/-7.6 ng/ml, respectively, before hCG injection and the concentration of estradiol decreased to 125.8+/-25.26 ng/ml, but concentration of progesterone increased to 195+/-24.6 ng/ml, 24h post-hCG injection. Inh-alpha and Cyp19A1 expressions in granulosa cells were maximal in the dominant follicle and declined in response to hCG treatment. Progesterone receptor, oxytocin and cycloxygenase-2 expressions in granulosa cells, regarded as markers of ovulation, were maximal at 24h post-hCG. The expressions of genes belonging to the super family of proteases were also examined; Cathepsin L expression decreased, while ADAMTS 3 and 5 expressions increased 24h post-hCG treatment. The results of the current study indicate that sequential treatments of PGF(2alpha) and hCG during early estrous cycle in the buffalo cow leads to follicular growth that culminates in ovulation. The model system reported in the present study would be valuable for examining temporo-spatial changes in the periovulatory follicle immediately before and after the onset of gonadotrophin surge.     

Apoptosis during spontaneous and prostaglandin F(2alpha)-induced luteal regression in the buffalo cow (Bubalus bubalis): involvement of mitogen-activated protein kinases

The present study was conducted to evaluate whether the corpus luteum (CL) of the water buffalo (Bubalus bubalis) cow undergoes luteal regression by the process of apoptosis and to examine the involvement of mitogen-activated protein (MAP) kinases during prostaglandin (PG) F(2alpha)-induced luteolysis. Sections of CL from late in the estrous cycle, i.e., during spontaneous luteolysis, stained for 4',6'-diamidino-2-phenylindole revealed increased numbers of condensed nuclei, indicating cell death by apoptosis, which was confirmed further by the occurrence of pronounced oligonucleosome formation. For morphological and biochemical characterization during PGF(2alpha)-induced apoptosis, CL were collected at 0, 4, 12, and 18 h after injection of 750 micro g of Tiaprost, a synthetic analogue of PGF(2alpha), to midestrous buffalo cows. Serum progesterone concentrations fell within 4 h and decreased (P < 0.05) maximally by 18 h. Concomitant decreases (P < 0.05) in the levels of steroidogenic acute regulatory mRNA and protein were observed in CL during 12-18 h, with the more profound effect on mRNA levels. Quantitative analysis of the genomic DNA showed a >5-fold increase (P < 0.05) in the low molecular weight DNA fragments by 18 h postinjection. Immunoblot analysis of CL tissue lysates showed increased (P < 0.05) levels of phospho-Jun N-terminal kinase (JNK) 1 (4- to 14-fold during 4-18 h) and phospho-p38 (2- to 4-fold at 18 h). Immunohistochemical evaluation of CL sections revealed an increased nuclear localization of phospho-JNK after treatment. These findings demonstrate that the CL of the buffalo cow undergoes cell death by the process of apoptosis both during spontaneous and PGF(2alpha)-induced luteolysis and that MAP kinases are involved during PGF(2alpha)-mediated apoptosis in the CL.     

Prostaglandin F2alpha-mediated activation of apoptotic signaling cascades in the corpus luteum during apoptosis: involvement of caspase-activated DNase

Prostaglandin F(2alpha) (PGF(2alpha)) acting via a G protein-coupled receptor has been shown to induce apoptosis in the corpus luteum of many species. Studies were carried out to characterize changes in the apoptotic signaling cascade(s) culminating in luteal tissue apoptosis during PGF(2alpha)-induced luteolysis in the bovine species in which initiation of apoptosis was demonstrable at 18 h after exogenous PGF(2alpha) treatment. An analysis of intrinsic arm of apoptotic signaling cascade elements revealed that PGF(2alpha) injection triggered increased ratio of Bax to Bcl-2 in the luteal tissue as early as 4 h posttreatment that remained elevated until 18 h. This increase was associated with the elevation in the active caspase-9 and -3 protein levels and activity (p < 0.05) at 4-12 h, but a spurt in the activity was seen only at 18 h posttreatment that could not be accounted for by the changes in the Bax/Bcl-2 ratio or changes in translocation of Bax to mitochondria. Examination of luteal tissue for FasL/Fas death receptor cascade revealed increased expression of FasL and Fas at 18 h accompanied by a significant (p < 0.05) induction in the caspase-8 activity and truncated Bid levels. Furthermore, intrabursal administration of specific caspase inhibitors, downstream to the extrinsic and intrinsic apoptotic signaling cascades, in a pseudopregnant rat model revealed a greater importance of extrinsic apoptotic signaling cascade in mediating luteal tissue apoptosis during PGF(2alpha) treatment. The DNase responsible for PGF(2alpha)-induced apoptotic DNA fragmentation was found to be Ca(2+)/Mg(2+)-dependent, temperature-sensitive DNase, and optimally active at neutral pH conditions. This putative DNase was inhibited by the recombinant inhibitor of caspase-activated DNase, and immunodepletion of caspase-activated DNase from luteal lysates abolished the observed DNA fragmentation activity. Together, these data demonstrate for the first time temporal and spatial changes in the apoptotic signaling cascades during PGF(2alpha)-in-duced apoptosis in the corpus luteum.     

Effects of bovine follicular fluid inhibin on serum gonadotrophin concentrations in ewes during oestrus

Experiments were conducted with ewes to investigate the effects of an enriched bovine follicular fluid inhibin preparation (INH) on gonadotrophin secretion after the onset of oestrus. Administration of INH (10 mg) 1 h after the onset of oestrus did not significantly alter the preovulatory FSH and LH surges or the second FSH peak. To determine the effects of INH on the second FSH surge, ewes were treated with saline (N = 7) or INH (N = 10) at 4 h (10 mg) and 24 h (5 mg) after the peak of the preovulatory LH surge. The second FSH surge was delayed about 24 h (P less than 0.05) in ewes treated with INH; however, the delay did not alter the interval to the next oestrus. In a third experiment, 16 ewes were assigned to 4 groups in a 2 x 2 factorial with the main effects being ovariectomy at 4 h and INH treatment (10 mg) at 4, 20 and 36 h after the peak of the LH surge. Controls received sham ovariectomy and saline injection as appropriate. Ovariectomy resulted in a rapid increase in serum FSH but not LH and this was delayed (P less than 0.05) by INH treatment. These results indicate that inhibin has a selective inhibitory action on FSH secretion in ewes and suggests that the second FSH surge results from increased basal FSH secretion due to decreased endogenous inhibin levels.     

The effect of progesterone replacement on gene expression in the corpus luteum during induced regression and late luteal phase in the bonnet monkey (<Emphasis Type="Italic">Macaca radiata</Emphasis>)

Background  

In higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles.     

Gene expression profiling of preovulatory follicle in the buffalo cow: effects of increased IGF-I concentration on periovulatory events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.     

Short-term fasting leads to inhibition of responsiveness to LH-stimulated testosterone secretion in the adult male bonnet monkey

A variety of stressors including fasting profoundly inhibit reproductive function in mammals. Although the effect of short-term fasting on gonadotropic axis is well established, the direct effects of fasting on gonads have not been reported. The objectives of the present experiments were to examine the effect of short-term fasting on circulating luteinizing hormone (LH) and testosterone (T) secretion, and to determine the responsiveness of testis to exogenous recombinant human (rh) LH treatment in male bonnet monkeys. In addition, an experiment was carried out to examine whether brief inhibition of endogenous LH secretion causes alteration in testicular responsiveness. Adult male monkeys were fasted for 1 day for examining the circulating endocrine hormone concentrations and challenged with rhLH injection 1 day after fasting. Food withdrawal for 1 day resulted in significant (P<0.05) decrease in LH, T and increase in cortisol concentrations. Surprisingly, T secretion in response to direct stimulation of Leydig cells by LH was not observed in fasted monkeys. In fed monkeys, treatment with Antide (a specific gonadotropin releasing hormone receptor antagonist to inhibit pituitary LH secretion) for 1 day did not compromise T secretion stimulated by rhLH, suggesting that loss of responsiveness of testis to exogenous LH treatment in fasted monkeys was not because of interruption in pituitary LH stimulation of the testis. The results indicate that short-term fasting in adult male monkeys cause inhibition of LH and T secretion, and inhibition of responsiveness of testis to LH stimulation.