Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy

The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.     

Expression of the uterine Mx protein in cyclic and pregnant cows,gilts, and mares

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.     

Plasma amino acid profiles at various reproductive stages in female rats

We measured the plasma levels of amino acids at various reproductive stages in female rats, including the estrous cycle, pregnancy and lactation, and compared the resulting amino acid profiles using two- or three-dimensional figures. These figures revealed that the amino acid profiles of pregnant and lactating dams differed considerably from those during the estrous cycle or in male rats. The plasma levels of individual amino acids were almost the same between proestrus, estrus, metestrus and diestrus, and their profiles did not differ significantly. However, the amino acid profiles changed during pregnancy and lactation in dams. The plasma Ser level decreased significantly in mid and late pregnancy, whereas Tyr, Gly and His decreased significantly in the late and end stages of pregnancy, and Trp and Lys significantly decreased and increased at the end of pregnancy, respectively. Much larger changes in amino acid profiles were observed during lactation, when the levels of many amino acids increased significantly, and none showed a significant decrease. Plasma Pro, Ser and Gly levels increased continuously from day 1 until day 15 of lactation, whereas Asn and Met increased significantly from days 1 and 5 respectively until the end of lactation. These results suggest that the profiles of plasma amino acids show characteristic changes according to reproductive stage and that it may be necessary to consider such differences when performing amino acid-based diagnosis.     

The effects of estrogen and progesterone on prostaglandins and integrin beta 3 (beta3) subunit expression in primary cultures of bovine endometrial cells

In cattle, endometrial expression of integrin alphavbeta3 is reduced on day 16 of the estrous cycle, coinciding with the critical period during which the decision is made to initiate luteolysis or continue with pregnancy. The objective of these experiments was to examine the relationship between estrogen and progesterone treatments, endometrial integrin alphavbeta3 expression, and prostaglandin F2alpha (PGF2alpha) and E2 (PGE2) production. Epithelial and stromal cells from intercaruncular (ICAR) and caruncular (CAR) bovine endometrium were treated with 17beta-estradiol (0.1 and 1.0 nM) and/or progesterone (1.0 and 10 nM) in a manner designed to mimic the steroid fluctuations of the estrous cycle. All cell types expressed estrogen receptor and progesterone receptor mRNA and protein. Intercaruncular stromal cells were the most responsive to steroidal regulation. Estrogen suppressed expression of integrin subunit beta3 mRNA in ICAR stromal cells (P< or =0.05). Progesterone and estrogen + progesterone treated cells did not differ in beta3 expression from controls (P> or =0.05). Steroid treatment did not affect PGF2alpha production in any cell type (P> or =0.05), however, estrogen decreased PGE2 production in all cells except CAR stroma (P< or =0.05). The results indicate that in bovine endometrium expression of integrin alphavbeta3 and production of PGE2 is influenced by estrogen.     

Excretion patterns of fecal progestagens, androgen and estrogens during pregnancy, parturition and postpartum in okapi (Okapia johnstoni)

The aim of the present study was to establish a simple method to monitor ovarian activity and non-invasively diagnose pregnancy in okapi (Okapia johnstoni). The feces of a female okapi were collected daily or every 3 days for 28 months. Steroids in lyophilized feces were extracted with 80% methanol, and the fecal levels of immunoreactive progestagens (progesterone and pregnanediol-glucuronide), androgen (testosterone), and estrogens (estradiol-17beta and estrone) were determined by enzyme immunoassays with commercially available antisera. Using the progesterone profiles, the durations of the luteal phase, follicular phase, and estrous cycle were determined to be 11.1 +/- 0.4, 5.3 +/- 0.6, and 16.5 +/- 0.7 days (n=22), respectively. Fecal levels of immunoreactive progesterone, pregnanediol glucuronide, and testosterone gradually increased from early pregnancy and peaked several months before parturition. More pregnanediol glucuronide was excreted in feces than progesterone during late pregnancy, but not during the estrous cycle. Although the fecal concentrations of immunoreactive estradiol-17beta and estrone change a little throughout pregnancy and non-pregnancy, they rose sharply and temporarily on the day following parturition. The present study indicates that fecal assays with commercial antisera for progesterone and pregnanediol glucuronide are useful for evaluating luteal activity and diagnosing pregnancy and indicates that estrogens might have some role as a trigger of parturition.     

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.     

Expression and Hormonal Regulation of Hoxa10 in Canine Uterus During the Peri-implantation Period

Hoxa10, a homeobox gene, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of Hoxa10 in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Hoxa10 mRNA was mainly localized in glandular epithelium and myometrium in canine uterus. There was a low level of Hoxa10 expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, Hoxa10 mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium. The expression of Hoxa10 mRNA gradually declined from day 23 and reached a low level on day 28. In the myometrium, a low level of Hoxa10 mRNA signal was seen on days 6, 12 and 17 of pregnancy and reached a high level on day 20 of pregnancy. During the estrous cycle, a high level of Hoxa10 mRNA expression was seen in the estrous uterus. Either estrogen or progesterone significantly induced the expression of Hoxa10 mRNA in the ovariectomized canine uterus. These results suggest that Hoxa10 expression is closely related to canine embryo implantation and upregulated by estrogen and progesterone.     

Effect of dietary energy restriction on metabolic and endocrine responses during the estrous cycle of the suckled beef cow

A replicated trial was conducted with suckled Angus and Polled Hereford cows (110 d postcalving) to determine metabolic and endocrine responses to an energy-restricted diet after cows had re-established postpartum estrous cyclicity. Cows were individually fed 26.5 Mcal ME (H) or 15.2 Mcal ME (L) for a 30-d preliminary period and fitted with an indwelling jugular cannula at synchronized estrus. Average daily weight change during the estrous cycle was .60 +/- .25 and -1.37 +/- .30 kg/d for H and L, respectively (P less than .05). Blood concentrations of cortisol, progesterone and LH during the estrous cycle were not affected by diet, nor did diet affect frequency or amplitude of LH pulses (P greater than .05). No dietary differences were observed for daily concentrations of total protein, glucose, nonesterified fatty acids or acetate. Mean blood concentrations of propionate and butyrate were not different between diets; however, L cows had lower concentrations of propionate and butyrate on d 11 of the cycle (P less than .05). Cows fed L had higher concentrations of blood urea nitrogen (P less than .05), but they had lower concentrations of cholesterol (P less than .05) on d 4, 11, 18 and subsequent estrus (E). Insulin was not different on d 4 and 11; however, cows fed L had lower insulin concentrations on d 18 and d E (P less than .05). Dietary energy restriction in these cyclic cows caused no change in endocrine responses. Of metabolic responses measured, only blood urea nitrogen, cholesterol and insulin showed consistent changes.     

Expressions of tumor necrosis factor-α, its receptor I,II and receptor-associated factor 2 in the porcine corpus luteum during the estrous cycle and early pregnancy

We examined the gene and protein levels of tumor necrosis factor (TNF)-α, its receptors (types I and II, designated TNF-RI and TNF-RII, respectively), TNF receptor-associated factor 2 (TRAF2) and morphological features in the porcine corpus luteum (CL), on Days 13 and 17 (Day 0 = the last day of estrus) of the estrous cycle or of early pregnancy. Gene expression levels of TNF-α, TNF-RI, TNF-RII and TRAF2 were unaffected by the day or reproductive status. TNF-α concentration was significantly higher in the CL on Day 17 of pregnancy than on Day 13 of pregnancy and on day 17 of the estrous cycle. The TNF-RI protein level was significantly higher in the CL on Days 13 and 17 of pregnancy than those of the estrous cycle, significantly increasing on Day 17 compared with those on Day 13 in pregnancy. In relation to TNF-RII protein levels, although there were no change during pregnancy, there was a tendency (P?=?0.0524) to up-regulate as pregnancy proceeded. In estrous cycle, TNF-RII protein levels decreased significantly as luteolysis proceeded. TRAF2 protein level was significantly higher in the CL on Days 13 and 17 of pregnancy than during estrous. There were few apoptotic bodies in the CL between Days 13 and 17 of pregnancy than during esrous. There were few apoptotic bodies in the CL between Days 13 and 17 of pregnancy. The number of apoptotic bodies was much greater than the CL on Day 17 of the estrous than those of pregnancy. Thus, the TNF-α and TNF-RI and TNF-RII pathways including the TRAF2 protein, known to control of cell differentiation, tissue renewal and apoptosis, might participate in maintaining the porcine CL during early pregnancy.     

Systemic progesterone concentration following human chorionic gonadotropin administration at various times during the estrous cycle in beef heifers

In Trial 1, 26 heifers were allotted randomly to a control group or one of four groups to receive a single injection of 3,000 IU hCG on d 1, 4, 7 or 10 of an estrous cycle. Heifers next completed a nontreated cycle, and at their third estrus were reallotted to one of the five groups described previously. Estrous cycle length was extended in cycle 1 but was not altered during the nontreated cycle or in cycle 3. Administration of hCG on d 4 or 7 increased (P less than .05) mean serum progesterone (P4) over the first 16 d of the estrous cycle by .9 and .8 ng/ml, respectively. In Trial 2, 23 heifers were allotted randomly to one of two groups to receive a placebo or a single injection of 3,000 IU hCG on d 4 of an estrous cycle. Heifers were inseminated artificially at subsequent estrus. On d 4 postbreeding, treatments were repeated. Administration of hCG on d 4 of the prebreeding estrous cycle increased (P less than .05) mean P4 over the first 16 d by .9 ng/ml, whereas mean P4 over the first 16 d postbreeding was not affected by a second injection of hCG on d 4 postbreeding. Administration of hCG increased (P less than .05) first-service pregnancy rate (92 vs 55%). In conclusion, progesterone concentrations were enhanced by hCG given on d 4 or 7 of the estrous cycle, and pregnancy rate was increased when hCG was administered both on d 4 of the prebreeding cycle and d 4 postmating.     

Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers

The objectives of the current study were to evaluate the correlation between reproductive status and steady-state levels of Myxovirus resistance gene (MX2) mRNA in peripheral blood leukocytes (PBL) of dairy heifers and the reliability of using change in MX2 messenger RNA (mRNA) for identification of nonpregnant heifers 18 to 19 d after AI. Holstein heifers (n = 266), 13 +/- 1 mo of age, were assigned randomly to be inseminated (BRED; n = 214) or not (NONBRED; n = 52). Estrous cycles of all heifers were synchronized with an intravaginal insert containing progesterone for 7 d. At insert removal, heifers received an injection of PGF2alpha. Heifers in the BRED group were inseminated on detection of estrus or at a fixed time, 72 h after insert removal concomitant with a GnRH treatment. Heifers in the NONBRED group received an injection of GnRH 48 h after insert removal. Blood samples collected on d 0 (d of AI or estrus) and 18 were used to determine steady-state levels of MX2 mRNA. Samples collected on d 0, 7, 14, and 21 were analyzed for progesterone concentration. Pregnancy was determined retrospectively by progesterone concentration on d 21 and was diagnosed at 30 +/- 1 and 60 +/- 3 d after AI. The fold change in levels of MX2 mRNA from d 0 to 18 was greater for heifers classified and diagnosed as pregnant on d 21 (P < 0.05) and 30 +/- 1 (P < 0.05) and 60 +/- 3 (P < 0.05) d after AI compared with nonpregnant (bred but not pregnant) and NONBRED heifers. Heifers that experienced pregnancy loss from 21 to 30 +/- 1 (P = 0.11) or 21 to 60 +/- 3 (P = 0.08) d of gestation tended to have smaller fold increases in MX2 mRNA expression than those that maintained pregnancy. The sensitivity (range 57.1 to 65.6%) and negative predictive values (range 47.9 to 57.1%) of determining reproductive status on d 18 according to the change in the level of MX2 mRNA expression in PBL were low, and the correlation between diagnosis of pregnancy by fold change in MX2 mRNA expression and other methods was small (r = 0.20 to 0.36). The current study indicates that increased expression of MX2 mRNA in PBL is related to pregnancy approximately 21, 30, and 60 d after AI in dairy heifers and that losses that occurred later in pregnancy were associated with lower fold increases in MX2 mRNA. However, using the change in MX2 mRNA expression was not a reliable method for diagnosis of pregnancy at 18 d after AI because of the low sensitivity and negative predictive value.     

Lamprey gonadotropin-releasing hormone-III selectively releases follicle stimulating hormone in the bovine

Recent studies have shown that lamprey gonadotropin-releasing hormone (l-GnRH) is localized in the mammalian brain, and that l-GnRH-III, can selectively induce FSH secretion in the rat both in vivo and in vitro. Consequently, the purpose of this study was to determine if l-GnRH-III could elicit selective FSH release in cattle and compare this response with that to mammalian luteinizing hormone releasing hormone (m-LHRH). Cattle were chosen as the animal model because previous studies have demonstrated that FSH and LH are secreted by separate gonadotropes in that species. For these studies, crossbred cycling heifers were implanted with jugular cannulae and l-GnRH-III was infused either between Days 9–14 or on Day 20 of the estrous cycle. Blood samples were collected both before and following peptide infusion. Our results demonstrate that during Days 9–14 of the estrous cycle (luteal phase), when progesterone levels averaged between 4 and 5 ng/ml, a dose of 0.25 mg of l-GnRH-III induced the release of FSH (P < 0.05), but not LH. A 0.5 mg dose of l-GnRH-III caused a greater release of FSH (P < 0.01), but still did not induce LH release. Higher doses of the peptide were capable of significantly releasing both gonadotropins. Importantly, during the luteal phase, doses of 0.5 and 2 mg of m-LHRH were ineffective in stimulating FSH, but did elicit marked increases (P < 0.001) in LH. Again, progesterone levels averaged 4–5 pg/ml. In order to assess gonadotropin releasing ability of l-GnRH-III at a different phase of the estrous cycle, some animals were administered the peptide on Day 20, when progesterone levels were below 1.0 pg/ml. At this time, the l-GnRH-III induced the release of LH (P < 0.01), but not FSH. Overall, our results demonstrate that l-GnRH-III can selectively induce FSH in cattle during the luteal phase, whereas m-LHRH was ineffective in that regard. Furthermore, the fact that l-GnRH-III can selectively stimulate FSH when serum progesterone is high, and LH when serum progesterone is low, suggests its actions are under strong control of this steroid. We suggest the FSH releasing capacity of l-GnRH-III in cattle could render this peptide useful for enhancement of reproductive efficiency in this species.     

Progesterone concentrations in beef heifers bred at puberty or third estrus

Peripheral serum progesterone concentrations were evaluated in beef heifers following breeding collected on d 6 +/- 1, 9 +/- 1 collected on d 6 +/- 1, 9 +/- 1 and 12 +/- 1 (estrus = d 0) after the puberal estrus of all heifers and after the third estrus of E3 heifers. Progesterone concentrations were higher (P less than .05) for heifers in E1 compared with heifers in E3 on d 6, 9 and 12 after breeding to a fertile bull. Progesterone concentrations on d 6, 9 and 12 did not differ (P greater than .10) between pregnant heifers in E1 and E3; however, non-pregnant heifers in E1 had higher (P less than .05) concentrations of progesterone compared with non-pregnant heifers in E3 on each day. Concentrations of progesterone did not differ (P greater than .10) between non-pregnant heifers in E1 and heifers of E3 during their puberal cycle. Pregnant heifers in E1 and E3 had higher (P less than .05) concentrations of progesterone on each day than non-pregnant heifers in their respective treatments. There were no interactions (P greater than .10) between treatment, pregnancy status and day-of-estrous cycle for concentrations of progesterone. Results of this study indicated that luteal function differed between heifers that failed to conceive at their puberal estrus and heifers that failed to conceive at third estrus. However, concentrations of progesterone did not differ between heifers that conceived at puberal or third estrus. The relationship of changes in luteal function from the puberal through the third estrous cycle and pregnancy is not clear.