Histological Characterization and Immunohistochemical Localization of Ki-67 and VEGF Factors in Bitches' Corpus Luteum at Cyclic and Early and Late Gestational Diestrous

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Localization patterns of steroid and luteinizing hormone receptors in the corpus luteum of Nelore (Bos taurus indicus) cows throughout the estrous cycle

The aim of the present study was to detect progesterone receptors (A and B isoforms), α and β estrogen receptors, luteinizing hormone receptors and aromatase cytochrome P450 enzymes in the corpus luteum of Nelore (Bos taurus indicus) cows using immunohistochemistry. The estrous cycles of 16 Nelore cows were synchronized, and luteal samples were collected via an incision into the vaginal vault. Samples were collected during specific days of the estrous cycle (days 6, 10, 15 and 18) and 24. h after circulating progesterone dropped, after luteolysis had occurred. After each biopsy was taken, all animals were resynchronized so that each biopsy was performed during a different estrous cycle. Our results showed that the concentration of studied proteins vary throughout the bovine estrous cycle. The highest concentration of α and β estrogen receptors and the highest concentration of plasma progesterone were both observed on days 10 and 15 of the estrous cycle. The highest concentration of progesterone receptors was observed on days 6 and 10 of the estrous cycle, and the most intense immunostaining for cytochrome P450 aromatase enzymes was observed on day 10 of the estrous cycle. The highest score of cells with plasma membrane immunostaining for LH receptors was observed on day 15 of the estrous cycle. In conclusion, this study demonstrates the varying concentrations of specific proteins within the corpus luteum of Nelore cows during the estrous cycle. This finding suggests that these receptors and enzymes, and their interactions, are important in regulating luteal viability. © 2013 Elsevier B.V.

Corpus Luteum Development and Function after Supplementation of Long-Acting Progesterone During the Early Luteal Phase in Beef Cattle

Contents: Strategic supplementation of P4 may be used to increase conception rates in cattle, but timing of supplementation in relation to ovulation, mass of supplementary P4 and formulation of the P4-containing supplement has not been determined for beef cattle. Effects of supplementation of long-acting progesterone (P4) on Days 2 or 3 post-ovulation on development, function and regression of corpus luteum (CL) were studied in beef cattle. Cows were synchronized with an oestradiol/P4-based protocol and treated with 150 or 300 mg of long-acting P4 on Day 2 or 3 post-ovulation (6-7 cows/group). Colour-doppler ultrasound scanning and blood sample collection were performed from Day 2-21.5. Plasma P4 concentrations were greater (p < 0.05) from Day 2.5-5.5 in the Day 2-treated groups and from Day 3.5-5.5 in the Day 3-treated cows than in the control group. CL area and blood flow during Day 2-8.5 did not differ (p > 0.05) among groups, suggesting no effect of P4 treatment on luteal development. The frequency of cows that began luteolysis before Day 15 was greater (p < 0.04) in cows treated with 300 mg than in the controls, but there were no differences between non-treated and 150 mg-treated cows. The interval from pre-treatment ovulation to functional and structural luteolysis was shorter (p < 0.01) in the combined P4-treated groups than in the control cows. In conclusion, was showed for the first time that long-acting P4 supplementation on Day 2 or 3 post-ovulation increases P4 concentrations for ≥3 day, has no effect on luteal development, but anticipates the beginning of luteolysis in beef cattle. © 2013 Blackwell Verlag GmbH.

Morphometric and Functional Characteristics of Buffaloes and Cows Corpus Luteum at Different Stages of the Estrous Cycle

The aims of the present study were to evaluate the morphometry of corpus luteum (CL) and progesterone (P-4) plasma concentration of 86 buffaloes (33 pregnant and 53 non-pregnant) and 95 cows (36 pregnant and 59 non-pregnant) at the moment of slaughter. Seventy CLs of buffaloes and 110 CL of cattle were analyzed. The CL classified as II and III were more common in both species (35.7 and 41.4% for buffaloes and 43.6 and 35.5% for cows). The 29 non-pregnant buffaloes had a total of 36 CL, being 19.4% CLI; 33.3% CL II; 27.8% CL III and 19.4% CL IV. The 51 nonpregnant cows had a total of 71 CL, being 26.8% CL I; 47.9% CL II; 21.1% CL III and 4.2% CL IV. The average diameters of bubaline and bovine CL were 5.2 +/- 0.9 and 6.4 +/- 1.8 mm (CL I); 17.6 +/- 2.6 and 19.8 +/- 3.2 mm (CL II); 17.2 +/- 2.1 and 20.0 +/- 3.2 mm (CL III); 7.8 +/- 1.8 and 8.7 +/- 2.7 mm (CL IV), respectively. The mean plasma concentrations of P4 were 5.6 (CL I); 5.4 (CL II); 4.7 (CL III) and 0.5 (CL IV) ng/mL for buffaloes and 0.02 (CL I); 6.3 (CL II) and 6.4 (CL III) ng/mL for cows. In both species, P4 concentration was similar between stages II and III. The results indicated that the characterization of the CL provides important information about the status of estrous cycle.

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold (P < 0.01) between days 13 and 22 of pregnancy and 8-fold (P < 0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").

In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin

Although it is well established that the secretory activity of the corpus luteum absolutely depends on the presence of pituitary-derived luteinizing hormone (LH), it is unknown why the life span of the corpus luteum is extended during early pregnancy by the placental production of chorionic gonadotropin (CG) but regresses in the presence of LH despite the fact that CG and LH have similar actions on the corpus luteum. To compare the responses of the corpus luteum to LH and human CG (hCG), cynomolgus monkeys whose endogenous gonadotropin secretion was blocked during the luteal phase of the menstrual cycle with a gonadotropin-releasing hormone antagonist were i.v. infused with either LH or CG. Infusion of LH at a constant rate overcame the gonadotropin-releasing hormone antagonist-mediated premature luteal regression but failed to prolong the functional life span of the corpus luteum. Continuous infusions of hCG did not effect a pregnancy-like pattern of gonadotropin secretion, but the functional life span of the corpus luteun was extended in two of three animals. Infusion of either LH or hCG in an exponentially increasing manner prolonged the functional life span of the corpus luteum beyond its normal duration. These results indicate that luteal regression at the termination of nonfertile menstrual cycles is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome by elevated concentrations of either LH or CG.

Toxicologia reprodutiva de ratos adultos expostos ao 2,3,7,8-Tetraclorodibenzo-P-Dioxina (TCDD) in útero

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First lactation ovarian function in dairy heifers in relation to prepubertal metabolic profiles

The aim of this study was to determine whether any differences in the GH-IGF-I axis in juvenile calves were predictive of fertility problems as adult cows. Endogenous metabolic hormone profiles before and after feeding and the response to a GH-releasing factor (GRF) challenge were measured in prepubertal (6 month) dairy calves. These metabolic parameters were subsequently related to physical characteristics at puberty and to ovarian function during the first lactation. Milk progesterone analysis was used to categorize the animals into those with normal progesterone profiles following calving (n = 17) and those that developed delayed ovulation (DOV1, n = 9) or persistent corpus luteum (PCL1, n = 6) profiles. There were associations between prepubertal GH parameters, glucose and non-esterified fatty acid (NEFA) concentrations and the body condition score at which the animals attained puberty. The calves which subsequently developed DOV1 profiles as cows tended to have a higher GH pulse amplitude during fasting than normal profile animals, they did not show the anticipated decrease in circulating glucose concentrations following a post-prandial rise in insulin and they also had the lowest IGF-I concentrations. The calves that later developed PCL1 had a significantly larger GH pulse amplitude and pulse area than normal profile animals in the fed period and had the highest IGF-I concentrations. There were no differences in prepubertal insulin or NEFA concentrations or in the GH response to a GRF challenge between the different progesterone profile categories. Plasma IGF-I concentrations in prepubertal animals were positively correlated with their post-calving concentrations, whereas glucose concentrations had a negative correlation between these time-periods. These results suggested that the different juvenile endocrine profiles of the DOV1 cows may predispose them to a higher rate of tissue mobilization during lactation and a consequent reduction in fertility, while altered GH and IGF-I levels in PCL1 cows may later contribute to the maintenance of the persistent corpus luteum. Therefore metabolic differences in prepubertal calves were later reflected by altered reproductive function during the first lactation.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.