Mohan Mondal

Deciphering the luteal transcriptome: potential mechanisms mediating stage-specific luteolytic response of the corpus luteum to prostaglandin F2α

The objective of this study was to identify prostaglandin F(2α) (PG)-induced changes in the transcriptome of bovine corpora lutea (CL) that are specific to mature, PG-responsive (day 11) CL vs. developing (day 4) CL, which do not undergo luteolysis in response to PG administration. CL were collected at 0, 4, and 24 h after PG injection on days 4 and 11 of the estrous cycle (n = 5 per day and time point), and microarray analysis was performed with GeneChip Bovine Genome Arrays. Data normalization was performed with affy package and significance testing with maanova from Bioconductor. Significance (relative to 0 h time point) was declared at fold change >2.0 or <0.5 and false discovery rate of <5%. At 4 and 24 h after PG, 221 (day 4) and 661 (day 11) and 248 (day 4) and 1,421 (day 11) regulated genes, respectively, were identified. The accentuated gene expression response in day 11 CL was accompanied by specific enrichment of PG-regulated genes in distinctive gene ontology categories (immune related and other), particularly at 24 h after injection. Specificity in putative transcription factor binding sites was observed among PG-regulated genes on day 11 vs. day 4, including a potential association of ETS transcription factors with acute PG-induced gene expression specific to day 11 CL. Temporal and PG-induced regulation of abundance of mRNA for ETS transcription factor family members linked to the stage-specific response to PG was not observed. Increased abundance of protein and/or mRNA for six PG-regulated putative ETS-responsive genes was noted in day 11 but not day 4 CL. Results reveal insight into stage-specific gene expression in bovine CL in response to PG and potential transcriptional mediators of luteolysis.

Regulation of Angiogenesis-Related Prostaglandin F2alpha-Induced Genes in the Bovine Corpus Luteum

ABSTRACT We recently compared prostaglandin F2alpha (PG)-induced global gene expression profiles in PG-refractory, bovine corpus luteum (CL) collected on Day 4 of the estrous cycle, versus PG-responsive, Day 11 CL. Transcriptome analyses led us to study the regulation of angiogenesis-related genes by PG and their functions in luteal endothelial cells (ECs). We found that PG regulated angiogenesis-modulating factors in a luteal stage-dependent way. A robust increase in FGF2 expression (mRNA and protein) occurred in the PG-refractory Day 4 CL promoting CL survival and function. Inhibitors of FGF2 action, thrombospondin 1 and 2, their receptor (CD36), and PTX3 were upregulated by PG specifically in Day 11 CL undergoing luteolysis. VEGF mRNA decreased 4 h post-PG in both Day 4 and Day 11 CL. The resulting destabilization of blood vessels in Day 11 CL is expected to weaken the gland and reduce its hormonal output. These genes were expressed in dispersed luteal ECs and steroidogenic cells; however, thrombospondin 1 and FGF2 were more abundant in luteal ECs. Expression of such genes and their ability to modulate FGF2 actions were investigated. Similar to its in vivo effect, PG, in vitro, stimulated the expression of thrombospondins and PTX3 genes in several luteal cell models. Importantly, these factors influenced the angiogenic properties of luteal ECs. FGF2 dose-dependently enhanced cell migration and proliferation, whereas thrombospondin 1 and PTX3 inhibited FGF2 actions in luteal ECs. Collectively, the data presented here suggest that, by tilting the balance between pro- and antiangiogenic factors, PG can potentially control the ability of the CL to resist or advance toward luteolysis.

Relationship of plasma estradiol-17β, total estrogen, and progesterone to estrus behavior in mithun (Bos frontalis) cows

The objectives of this study were (1) to establish the characteristics of estrus behavior in mithun cows (n = 12) and (2) to determine the relationships between this behavior and the plasma concentrations of estradiol-17beta (E2), total estrogen, and progesterone. Estrus was detected by visual observations of estrus signs, per recta examination of genitalia and bull parading thrice a day for three consecutive cycles. Among the behavioral signs of estrus, the cow to be mounted by bull (100%) was the best indicator of estrus followed by standing to be mounted (92%). Per rectum examination of genital organs revealed relaxed and open os externa of cervix, turgid uterus, and ovaries having palpable follicles in all animals. The mean (+/-SEM) length of estrus cycle and duration of estrus were recorded to be 21.8 +/- 0.69 days and 12.6 +/- 1.34 h, respectively. Endocrine profiles during the peri-estrus period showed that the mean highest peak concentrations of E2 (27.29 +/- 0.79 pg/ml) and total estrogen (45.69 +/- 2.32 pg/ml) occurred at -3.90 +/- 2.27 and -3.89 +/- 2.26 h prior to the onset of estrus, respectively. Plasma progesterone concentration was basal (0.14 +/- 0.001 ng/ml) during the peri-estrus period. Plasma E2 and total estrogen were found to increase from 6 days before estrus to reach a peak level on the day of estrus and decline thereafter to basal level on day 3 of the cycle. The plasma progesterone concentration was the lowest on the day of estrus showing gradual increase to register a peak level on day 15 of the cycle. Estrus behavior was found to be positively correlated with the maximum peak concentration of E2 (r = 0.89; P < 0.0001) and total estrogen (r = 0.66; P = 0.019) during the peri-estrus period. The mean total estrogen concentration during the peri-estrus period was significantly correlated with estrus behavior (r = 0.60; P = 0.04). The correlations between the estrus behavior and E2:progesterone ratios at 6 days before the onset of estrus (r = 0.92) and on the day of estrus (r = 0.95) was significant. The total estrogen:progesterone ratios at 6 days before the onset of estrus and on the day of estrus were also positively correlated with the estrus behavior (r = 0.86 and 0.88). In conclusion, our results suggest that the maximum peak concentration of E2 and total estrogen and mean level of total estrogen during the peri-estrus period and the E2:progesterone and total estrogen:progesterone ratios on 6 days before the onset of estrus and on the day of estrus are the important factors contributing the behavioral manifestation of estrus in mithun cows.

Evidence Supporting a Role for Cocaine- and Amphetamine-Regulated Transcript (CARTPT) in Control of Granulosa Cell Estradiol Production Associated with Dominant Follicle Selection in Cattle

We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.

Secretion Patterns of Growth Hormone in Growing Captive Mithuns (Bos frontalis)

Abstract A study was conducted in May 2003 to characterize plasma growth hormone (GH) pattern in growing mithuns (Bos frontalis), a rare semi-wild ruminant. Six mithun calves averaging 235 day of age and 124 kg were maintained in semi-intensive system and group-fed once daily. Animals gained at a mean rate of 0.54 kg/day, with individuals ranging from 0.34 to 0.66 kg/day. Blood samples collected at 15-minute intervals starting from 0600h for nine-hour period were assayed for plasma GH. Growth hormone patterns consisted of frequent pulses of varying amplitude. Growth hormone pulses occurred at an average frequency of 0.69/h, the rate did not differ markedly among mithuns nor hour of day. The magnitude of GH secretory pulses varied significantly among mithuns. Growth hormone peaks averaged 95.0 and 45.2 ng/ml in mithuns having the highest and lowest GH peaks, respectively. Peak and mean GH levels were associated positively (r=0.98, P<0.001) and both were associated negatively (r=−0.97 and −0.98, respectively; P<0.01) with rates of gain. Results from the study show that 1) GH peaks occur at frequent intervals throughout the sampling period and 2) alteration in GH levels and patterns are elicited more by pulse amplitude than frequency modulation.

Development and validation of a simple sensitive enzyme immunoassay (EIA) for GH determination in buffalo plasma.

Abstract A simple and highly sensitive enzymeimmunoassay (EIA) for GH determination in buffalo plasma on microtitreplates using biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 100 µL buffalo plasma. The GH standards ranging from 0.05 ng/well/100 µL to 12.8 ng/well/100 µL were prepared in hormone free plasma collected from an aged (>15 years) senile buffalo. The sensitivity of the EIA procedure was 50 pg/well GH, which corresponded to 0.50 ng/mL plasma; the 50% relative binding sensitivity was seen at 800 pg/well/100 µL. Plasma volumes for the EIA, viz., 25, 50, and 100 µL did not influence the shape of standard curve, even though a slight drop in the OD450 was seen with higher plasma volumes. For the biological validation of the assay, 12 Murrah buffalo calves were used. Six of these were administered synthetic bovine growth hormone-releasing factor (10 µg/100 kg body weight, i.v., and the remaining six animals were administered sterile normal saline and kept as controls. Jugular blood samples were collected at −60, −45, −30, −15, −10, −5, 5, 10, 15, and 30 min and, thereafter, at an interval of 15 min using an indwelling jugular catheter, beginning 1h prior to GRF injection up to 8 h post treatment. In all animals, a peak of GH was recorded within 5 to 20 min of GRF administration, which confirms the biological validation of the EIA. To confirm homogeneity of buffalo GH with bovine GH, a parallelism test was conducted between the buffer standard curve of bovine GH and GH measured from serial dilution of buffalo plasma containing a high level of endogenous growth hormone.

Development and Validation of a Simple, Sensitive, Second Antibody Format Enzyme Immunoassay (EIA) for LH Determination in Mithun (Bos Frontalis) Plasma

Abstract The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin‐streptavidin amplification system and the second antibody coating technique. Biotin was coupled to LH and used to bridge between streptavidin‐peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20 µL mithun plasma. The LH standards ranging from 6.25 pg/well/20 µL to 400 pg/well/20 µL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100 pg/well/20 µL. Plasma volumes for the EIA viz. 10 and 20 µL did not influence the shape of standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10 µg i.v., with a synthetic analogue of GnRH (Buserelin‐Acetate, Intervet, India) and blood samples were collected at 15 min intervals using indwelling jugular catheter beginning 1 h prior to GnRH injection till 8 h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun.

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

Brilliant cresyl blue (BCB) is a super‐vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocytes developmental potential, and the transcript abundance for select TGFβ‐superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus‐cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low‐quality oocytes, based on BCB staining, was also determined. Cumulus‐oocyte complexes (COCs) from abattoir‐derived ovaries were subjected to BCB staining, and germinal‐vesicle‐stage oocytes and cumulus cells were harvested from control, BCB+, and BCB− (low‐quality oocyte) groups for real‐time PCR or Western‐blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB− oocytes. Western‐blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB− oocytes. Embryo‐culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY‐1 treatment, improve the developmental competence of BCB− oocytes. These results contribute to a better understanding of molecular indices of oocyte competence. Mol. Reprod. Dev. 82: 251–264, 2015.

Characterization of Mithun (Bos frontalis) ejaculates and fertility of cryopreserved sperm

Technologies for conservation and propagation of genetic resources in the Mithun (Bos frontalis), a rare semi-wild bovine species of Southeast Asia. Successful cryopreservation of Mithun semen would provide a potential vehicle to address above issue. To date, information on characteristics of Mithun ejaculates is not available and there are no reports of birth of live offspring using cryopreserved Mithun semen collected using AV method. A study was therefore conducted to (i) characterize the Mithun ejaculate, (ii) investigate the effectiveness of Mithun sperm cryopreservation, and (iii) determine whether artificial insemination using frozen-thawed Mithun sperm can result in live offspring. Semen samples collected from eight fertile Mithun bulls were evaluated for colour, consistency, volume, concentration, mass activity and progressive motility. The freshly ejaculated sperm were also evaluated for morphological abnormalities, live sperm counts, acrosome integrity, membrane stability (hypo-osmotic swelling test; HOST) and DNA integrity. Semen samples of good quality were cryopreserved in liquid nitrogen using Tris-egg yolk-glycerol diluent. Post-thaw quality of the cryopreserved sperm in terms of progressive motility, morphological abnormalities, live sperm counts, acrosome integrity, membrane stability and DNA integrity were assessed. In addition, 16 Mithun cows at estrus were inseminated with frozen-thawed Mithun sperm. Following cryopreservation, the percentage of progressive motility (fresh versus frozen-thawed), live sperm counts, morphological abnormalities, acrosome integrity, membrane stability and DNA integrity were found to decrease (P<0.01) with a motility recovery rate of 74+/-9%. Mithun cows inseminated with cryopreserved sperm result 75% conception rate and all the conceived cows maintained full-term pregnancy with delivery of live calves.

Relationship between plasma growth hormone concentrations and temperament in mithuns (Bos frontalis)

The aim of the present study was to verify whether or not plasma growth hormone (GH) concentrations are correlated with temperament in mithuns (Bos frontalis), a semiwild ruminant. Therefore, a total of 69 female mithuns from four different strains, viz., Arunachal, Nagaland, Mizoram, and Manipur, were divided into six age groups (Group I, 0-6 months; Group II, >6-12 months; Group III, >1-2 years; Group IV, >2-2.5 years; Group V, >2.5-3.0 years; Group VI, >3.0 years). Blood samples were collected weekly for 6 consecutive weeks and assayed for plasma GH. Temperament was scored on a 6-point scale, 6 were being very aggressive and 1 docile. Body weights of all animals were recorded once a week for 6 consecutive weeks GH concentrations and temperament scores were found to differ significantly between groups. Strain had significant effects on blood GH levels and temperament. Blood GH concentrations and temperament of Manipur mithuns were significantly higher than those of the other three strains within each group, for all groups. Across groups III to VI, blood GH levels and temperament among Nagaland, Mizoram, and Arunachal mithuns did not differ. Overall, the strain with the highest blood GH concentrations also had highest temperament scores. The Manipur strain had the highest blood GH levels and exhibited the most aggression (r = 0.95), and Arunachal mithuns, the lowest (r = 0.93). Temperament scores tended to decrease with increasing age for all four strains. Coefficients of correlation between blood GH and temperament among strains within each group, for all groups, were found to be significant. The highly positive correlation (r = 0.94) between blood GH concentrations and temperament for all animals, regardless of age and strain differences, clearly indicates the relationship between blood GH and temperament in mithuns. In conclusion, our results suggest that peripheral blood GH levels can influence temperament in mithuns.