Changes in the expression of toll-like receptor mRNAs during follicular growth and in response to lipopolysaccharide in the ovarian follicles of laying hens

The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.     

Morphologic changes in the ovary and histologic changes in the tertiary ovarian follicles in ewes after prolonged irradiation during anestrus

The changes in volume, weight and the histomorphological changes of the tertiary follicles of ewes were studied after protracted irradiation with 4.8 Gy in the anoestrous period by the morphometric and qualitative histological methods. The trial was performed in May with 21 ewes of the Slovak Merino breed, divided into three groups. The first group (five ewes) was control. The second and third groups (each containing eight ewes) were exposed to gamma-rays for five days, the total dose being 4.8 Gy. Within ten days after the treatment, all the irradiated and control ewes were given Ampicillin Spofa per os at a dose of 250 mg per head/day and Roboran Spofa at a dose of 10 g per head/day. The animals were killed by bleeding on the fifth day of irradiation and on the tenth day after the end of the treatment. After killing, the volume and weight of the ovaries were determined and a common histological method was used to cut these ovaries into 7 microns slices in series 70 microns apart. The slices were stained with haematoxylin-eosine and were evaluated by means of light microscopy. After irradiation the weight of the ovaries was found to decrease significantly; however, ovary volume remained unchanged. The atretic and non-atretic tertiary follicles were subjected to qualitative histological differentiation after Marion et al. (1968) and the number of non-atretic follicles was found to have decreased significantly in the irradiated ewes. The late type of atresia contributes most significantly to an increase in the proportion of atretic tertiary follicles. The administration of vitamins after irradiation reduced the occurrence of atretic changes.     

Use of melengestrol acetate and gonadotropins to induce fertile estrus in seasonally anestrous ewes.

Ewes of three genotypes (Hampshire, n = 59; Rambouillet, n = 36; crossbred, n = 57) were used to determine the efficiency of melengestrol acetate (MGA) and(or) PG-600 (a combination of pregnant mare's serum gonadotropin and human chorionic gonadotropin) in inducing fertile estrus in seasonally anestrus ewes. Ewes were assigned randomly, within genotype, to treatments in a 2 x 2 factorial arrangement. Treatments were control, .125 mg of MGA given twice per day for 9 d (MGA), a single 5-mL injection of PG-600 (PG-600), and the combination of treatments MGA and PG-600 (MGA/PG-600). Feeding of MGA began on May 14, 1990, and ended on May 23. Injections of PG-600 were given immediately after the last feeding of MGA or vehicle on May 23. All ewes were exposed to fertile, brisket-painted rams on May 24 (d 0) for 40 d. Ewes were checked for estrus twice daily for 9 d. Laparoscopy was performed, to assess ovulation rate (OR), on d 6 for ewes that were not detected in estrus and on d 12 for ewes that exhibited estrus. Percentage of ewes mated was increased by MGA (P less than .001). Ovulation rate of ewes exposed to rams was increased by PG-600 (P less than .01) and this effect was enhanced by MGA (P less than .05), whereas MGA alone tended to decrease OR (P less than .10). Melengestrol acetate decreased the interval to lambing by 6.5 d (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen receptors in the uterus and ovarian follicles of gilts treated with dihydrotestosterone

Experiments were conducted to evaluate expression of the estrogen receptor (ER) alpha and ERbeta genes in the uterus and ovarian follicles of gilts treated with 5alpha-dihydrotestosterone (DHT) during the follicular phase of the estrous cycle. This DHT treatment has enhanced ovulation rate but decreased blastocyst survival in previous experiments. Gilts received daily i.m. injections of 10 mg of DHT from day 13 (day 0 = onset of estrus) to day 18 (experiment 1), or from day 13 to 16 (experiment 2) of the estrous cycle. Gilts that served as controls received vehicle. The ovaries and a portion of uterine horn were surgically removed 24 h after the last treatment. Administration of DHT from day 13 to 18 of the estrous cycle decreased uterine wet weight (tendency, P = 0.10), and the relative amounts (ratios to ribosomal protein L19) of endometrial mRNA for the estrogen-responsive gene complement component C3. Gilts receiving DHT had greater amounts of ERbeta mRNA in the endometrium than those treated with vehicle in both experiments, but DHT did not alter the overall amounts of endometrial ERalpha mRNA. Immunohistochemical (IHC) analysis demonstrated that DHT did not alter the relative amounts of ERalpha in the myometrium, glandular and luminal epithelia and endometrial subepithelial stroma. In the ovary, amounts of ERalpha and ERbeta mRNAs in surface walls of follicles > or =6 mm in diameter were not altered by DHT treatments, however, DHT treatment from day 13 to 16 decreased the amounts of immunoreactive ERalpha in the theca interna at the surface walls of day 17 follicles (experiment 2). The amounts of immunoreactive ERalpha were greater in the granulosa than in the theca interna, and within cell type, the amounts of ERalpha were greater at the surface than at the basal region of the follicles, with the exception of the theca interna in follicles evaluated on day 19 (experiment 1). Treatment of gilts with DHT during the follicular phase of the estrous cycle increased ERbeta mRNA in the endometrium and influenced the amounts of immunoreactive ERalpha in ovarian follicles in a cell type-, day of development- and region-specific manner.     

Changes in the follicle system and the population of tertiary follicles in sheep after the administration of PMSG in anestrus

The total quantitative changes of ovaries, proportion of atretic and non atretic follicles and changes of tertiary follicles in sheep after administration of increasing doses of PMSG during the anoestrous period were observed. In experimental groups the statistically significant increase of average weight, volume and dimensions of ovaries in comparison with control group were determined biometrically. The average number of tertiary follicles was greater in experimental groups but at the same time we observed a higher proportion of atretic follicles (64% of the total number in the control group; 71-77% in the experimental groups). In the group of sheep administered a dose of 1500 m.u. PMSG we determined a high proportion of luteinized follicles (as much as 21% of the total number of atretic follicles). The total number of small follicles in the so called transient phase in the comparison of experimental and control groups was not changed significantly. In the experimental group an increased incidence of preovulatory follicles and a reduction of tertiary follicle dimensions in the period of follicle cavity formation was determined.     

Inhibin and reproduction. First communication: changes to inhibin level in Barki ewes after artificial infection with bluetongue virus

Bluetongue virus type 16, isolated from sheep in Egypt, was injected to 4 normally cycling Barki ewes and caused high levels of inhibin. This was assayed by a biological method, using suppression of the luteinising hormone (LH) of castrated rats. Albumin (fraction 2) was injected to normally cycling ewes. The sera of injected ewes were investigated 1 day after injection and weekly up to the 4th one injection. There was a gradual decrease of LH (4.8 +/- 0.52 I.U./ml serum) until the minimum level (1.17 +/- 0.25 I.U./ml serum) was reached, in comparison to the control serum LH which was 5.26 +/- 0.52 I.U./ml serum during the dioestrous phase.     

Control of ovarian follicles activity in the ewe.

During the ovine estrous cycles, three waves of follicular growth, closely associated with the FSH secretion pattern, were observed. The parameters of these follicular waves and the ability of follicles to produce steroids in vitro were studied in various conditions. In vivo, the follicular events were similar between the breeding season and the anestrus, except for the lack of ovulation; but at the end of the breeding season and in anestrus, the follicles lose a big part of their aromatization ability. In ewes carrying the Booroola fecundity gene or Cambridge fecundity gene, the reduction in follicular atresia seems to be one of the main follicular features implicated in the control of high ovulation rate. In vitro, the most relevant difference is an early acquisition of estrogen production ability of small follicles in Booroola fecundity gene barring ewes. Fluoro-gestone-acetate (FGA) pessaries reduced the number of growing follicles; despite this effect disappearing after the sponge withdrawal, the ovulation rate is significantly reduced. But an equine chorionic gonadotrophin (eCG) treatment restores the ovulation rate (OR) by reducing the atresia rate of pre-ovulatory follicles. In similar conditions, a pretreatment of the ewes with melatonin again reduced the atresia rate of large follicles and resulted in an increased ovulation rate. In vitro, FGA blocked aromatization ability, and melatonin inhibited both androstenedione and estradiol production, but a further treatment with eCG partly restores the steroid secretion. Immunization against androstenedione leads to a higher OR, owning to a reduced atresia of large follicles. Daily growth hormone injections for a hole cycle resulted in an increased follicular population and ovulation rate, while FSH plasma levels decreased and the follicle sensitivity to gonadotrophins was reduced.     

Changes in the concentration of hypothalamic and hypophyseal receptors for estradiol in pregnant and postpartum ewes

The sensitivity of the hypothalamic-pituitary axis to estradiol was evaluated by monitoring changes in the concentration of receptors for estradiol during gestation and the postpartum period of the ewe. Ewes that had been bred during anestrus so that lambing would occur during the breeding season were killed on d 50 and 140 of gestation or on d 2, 13, 22 and 35 postpartum. Total cytosolic receptors for estradiol were quantified in the anterior and posterior hypothalamus, median eminence, anterior pituitary and cerebral cortex. Concentration and total number of receptors for estradiol in the anterior and posterior hypothalamus and anterior pituitary gland during gestation through d 13 postpartum were similar but increased (P less than .05) by d 22 and 35 postpartum. Changes in the concentration of receptors for estradiol were tissue-specific because stage of reproduction did not influence the number of receptors for estradiol in the median eminence or cerebral cortex. Increases in the concentration of receptors for estradiol in hypothalamic and pituitary tissues on d 22 postpartum immediately preceded the onset of normal ovarian cyclicity in ewes. From these data we conclude that resumption of reproductive cycles in postpartum ewes occurs about the time concentrations of receptors for estradiol in the anterior pituitary gland and hypothalamus increase.     

A study on the effect of GnRH administration on the ovarian response and laparoscopic intrauterine insemination of Awassi ewes treated with eCG to induce superovulation

The effect of GnRH administration on superovulatory response of ewes treated with equine chorionic gonadotrophin (eCG) in breeding and nonbreeding seasons and the contribution of laparoscopic insemination to the improvement of fertilization and embryo recovery were investigated. Twenty-four nonpregnant Awassi ewes of 3–4 years of age were randomly allocated into two groups (n = 12). Each ewe was treated with a progesterone impregnated intravaginal sponge for 12 days. The following superovulation treatment was used: ewes of group 1 received 1,200 IU of eCG once as an intramuscular injection 48 h prior to sponge withdrawal; ewes of group 2 also received 1,200 IU of eCG once as an intramuscular injection, 48 h prior to sponge withdrawal and after 24 h of sponge removal. Ewes were injected with 80 μg of GnRH. Ewes of groups 1 and 2 were further subdivided into four equal groups (n = 6). Subgroups A and C (superovulated with eCG and eCG plus GnRH, respectively) were mated naturally at least two times with Awassi rams of proven fertility at 8-h intervals. Subgroups B and D (same as A and C) had intrauterine insemination at 44–46 h after sponge removal, under laparoscopic visualization of uterine horns, depositing 1 ml of diluted semen containing 100 × 10⁶ motile sperm in the distal portion of each uterine horn. Ovarian response was assessed by determining the number of corpora lutea by laparoscopy at day 6 after mating. Embryo recovery was performed by using a semi-laparoscopic flushing procedure in both uterine horns. Results of the present study showed that ewes treated in breeding season with eCG plus GnRH has a higher number (P < 0.05) of corpora lutea than eCG alone as 7.33 ± 0.54 and 4.33 ± 0.39, respectively. There was no significant difference in the number of corpora lutea in nonbreeding season when ewes treated with eCG and eCG plus GnRH. The number of unovulated follicles was significantly higher (P < 0.05) in eCG treated ewes than in ewes treated with eCG plus GnRH, both in the breeding and nonbreeding seasons. The number of recovered embryos from ewes treated with eCG plus GnRH and eCG differ significantly (P < 0.05) as 4.32 ± 0.56 and 1.06 ± 0.26, respectively, in the breeding seasons. No significant difference was observed when these hormones used for superovulation in the nonbreeding season. A higher number of unfertilized ova (P < 0.05) was observed in ewes when naturally inseminated than in ewes inseminated using the intrauterine laparoscopic technique. Higher rate of embryo recovery (P < 0.05) was achieved when ewes were inseminated via intrauterine (4.66 ± 0.66) compared with ewes naturally mated (2.16 ± 0.74). The fertilization rate in ewes inseminated intrauterine using laparoscopic techniques and naturally mated were 91.5% and 44.8%, respectively. Fertilization failure in ewes inseminated intrauterine using laparoscopic techniques and naturally mated were 8.4% and 55.2%, respectively. It could be concluded that administration of GnRH 24 h after sponge removal increased ovulation rate of Awassi ewes treated with eCG for superovulation in the breeding season. The use of eCG to induce superovulation in Awassi ewes combined with laparoscopic intrauterine insemination increases the fertilization rate.     

Changes in reactivity to halothane after administration of mephenytoin

In the pigs of the Belgian Landrace breed, peroral premedication removed sensitivity to halothane. When mephenytoine had been administered four hours before testing (dose of 4 mg/kg live weight), the sensitive animals ceased to differ in their reaction to halothane from non-sensitive animals.     

Changes in surface follicles and ovarian weight in testing for responsiveness in dairy cows after induction of superovulation using PMSG and cloprostenol

We evaluated the ovaries of 34 cows cross-bred of the Slovak Pied and Lowland Black-pied breeds which were culled and intended for slaughter during the winter type of feed rations. For superovulation treatment we used PMSG in the preparation Serum Gonadotropin (Bioveta, Nat. Ent., Ivanovice na Hané) and cloprostenol in the preparation Oestrophan inj. Spofa. We weighed the excised ovaries, fixed them in formalin 10% and made a quantitative evaluation of the surface follicles and differentiated them into recruited and selected or dominant follicles. We determined the concentration of 17 beta-estradiol and testosterone with the aid of the 3H RIA set from the firm Sorin after extraction by diethylether with separation of free and bound hormone with active charcoal. 72 hours after the giving of cloprostenol 43% reacted positively to superovulation treatment and after seven days a 50% positive response was recorded. After a dose of 2000 i. u. of PMSG (n = 6) embryo was obtained, whereas after 3000 i. u. of PMSG (n = 8) we flushed out eight embryos, of which four zygotes were suitable for transfer. After a higher dosage of PMSG there was an increase in the average weight of the ovaries, in right-hand ovaries significantly with P less than 0.05. After super-ovulation treatment the concentration of 17 beta-estradiol (E2) in the follicular fluid from follicles seven days after insemination was found to be so low as to be below the limit of detection, with the exception of four samples (mean = 8.60 nmol.l-1). The greatest concentration of E2 was from animals (n = 5) 72 hours after the giving of cloprostenol--1099.61 nmol.l-1) of follicular fluid. The concentration of testosterone was lower in the follicles of untreated cows in the follicular phase (mean = 5.92 nmol.l-1) compared with the follicles of super-ovulated animals the seventh day after insemination (mean = 14.12 nmol.l-1). The number of recruited and especially selected surface antral follicles 72 hours (n = 7) after the giving of cloprostenol and seven days (n = 8) after insemination was significantly higher in the group of brood cows reacting positively to superovulation in comparison with the animals which did not respond. It appears that the simultaneous monitoring of hormonal and morphological changes in the follicular system will help in objectivising the evaluation of the functional activity of stimulated follicles.     

Immunohistochemical localization of inhibin subunits in the testis of the bull

The differential localization of the inhibin beta subunits betaA and betaB in the testis of adult bull was studied using specific monoclonal and polyclonal primary antibodies. Inhibin betaA- and betaB-subunits were localized only in the Sertoli cells. The inhibin betaA-subunit was observed in the cytoplasm while the betaB-subunit was localized in the nucleus. No specific findings depending on spermatogenic stages were observed among the seminiferous tubules. Moreover, the inhibin alpha-subunit was not detected in the testis of the bulls. In addition, no inhibin subunits were detected in the Leydig cells and spermatogenic cells. These findings indicate the presence of betaA- and betaB-subunits in the bull, which may suggest a possibility that activin is produced and/or stored in the Sertoli cells and regulates spermatogenesis in an autocrine/paracrine manner. Moreover, the inhibin betaB-subunit may be produced in the nucleus but the functional meaning of this is not yet clear.     

Effect of large palpable ovarian follicles on response to prostaglandin administration in dairy cows with corpora lutea

We examined the response to exogenous prostaglandin F2α in cattle with or without palpable structures believed to be ovarian follicles. All animals had ovarian structures diagnosed by palpation as corpora lutea. The cows were placed into two groups: those with follicles which were estimated by the palpators to be ≤13 mm diameter (n=60); and cows with no palpable follicles or with follicles <13 mm diameter (n=133). Comparisons of proportion in estrus within five days, days to estrus, and milk progesterone levels failed to show significant differences between the groups.     

Effects of sodium pentobarbital on release of gonadotropins in ewes immediately after ovariectomy and after treatment with gonadotropin-releas ing hormone

Two experiments were conducted with ewes 9 to 11 days after estrus to determine whether the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are controlled differentially. In experiment 1, gonadotropin-releasing hormone (GnRH) was injected (100 (μg/ewe) at time = 0 min into ewes in four treatment groups. The treatment groups (9 ewes/group) were: 1) periodic iv sodium pentobarbital (NaPen) vehicle from 0 min; 2) periodic iv NaPen from 0 min; 3) vehicle iv for 120 min then iv NaPen from 120 min; 4) vehicle iv for 150 min then iv NaPen from 150 min. A surgical plane of anesthesia was maintained from the initiation of NaPen injection until the experiment ended. Jugular blood was sampled at 30-min intervals from ?30 to + 210 min for LH and FSH assays, and profiles of hormone concentrations were compared by time-trend analyses. GnRH released LH (P<.001) and FSH (P<.001), but NaPen did not affect the profiles of hormone concentrations; this indicated that NaPen did not reduce the ability of the pituitary to secrete gonadotropins in response to GnRH. Experiment 2 was a 2x2 factorial with ovariectomy (time = 0 hr) and NaPen as the main effects. One group of ovariectomized (n = 6) and one group of sham ovariectomized (n = 6) ewes were anesthetized only during surgery, while a group of ovariectomized (n = 7) and a group of sham ovariectomized (n = 6) ewes were kept at a surgical plane of anesthesia until 10 hr after surgery. Patterns of LH and FSH were compared in jugular blood collected hourly from 0 hr until 10 hr after surgery and in samples collected at 24 hr intervals from -24 to +72 hr of surgery. After ovariectomy, LH increased (P<.001) hourly and daily, but anesthesia suppressed (hourly, <.001 and daily, P<.005) these increases, which resulted in an interaction (hourly, P<.001 and daily, P<.01) of ovariectomy and anesthesia. FSH after ovariectomy increased hourly and daily (hourly, P<.02 and daily, P<.001), but the effect of anesthesia and interaction of ovariectomy and anesthesia were not significant. Because NaPen did not alter secretion of LH or FSH after exogenous GnRH in experiment 1 while it blocked the postovariectomy increase in LH but not FSH in experiment 2, we concluded that the postovariectomy increase in LH resulted from increased hypothalamic secretion of GnRH. The mechanisms responsible for the postovariectomy increase in FSH secretion are not identical to those for LH. The mechanisms that control the postovariectomy secretion of FSH might involve factors that are not suppressible by NaPen or, alternatively, the differences in LH and FSH release after ovariectomy might reflect the removal of ovarian factors that suppress FSH but not LH secretion in intact ewes.     

A role for the adrenal cortex in the onset of cystic ovarian follicles in the sow.

The corticotrophin (ACTH)-adrenal cortical axis has previously been implicated in the onset of cystic ovaries in the sow. In view of the role of the ACTH-adrenal cortical axis in stress, two sows were subjected to an elevated environmental temperature of 32 degrees C for three hours daily during the follicular phase of the estrous cycle. Plasma concentrations of glucocorticosteroids and progesterone fluctuated markedly in one sow that developed cystic ovaries. Concentrations of these hormones did not vary greatly in the other sow that did not develop cystic follicles. Exposure to an environmental temperature of 32 degrees C for three hours or injection of 1 IU/kg bodyweight of ACTH for each of two ovariectomized sows resulted in an elevation in progesterone values to 5-7 ng/ml plasma from basal levels of 1-2 ng/ml and a rise in total glucocorticosteroids from basal levels of 1 or 2 microgram/100 ml plasma to 4-10 microgram/100 ml. Injection of 2 mg/kg bodyweight of progesterone and 4 mg/kg bodyweight of cortisol into the ovariectomized sows was found to approximate these elevations in plasma steroid values. When either progesterone or cortisol was injected daily during the follicular phase into two intact sows in two successive experiments at these dosage levels, similarly elevated plasma steroid concentrations were seen and cystic ovarian follicles resulted. The results suggest that glucocorticosteroids and progesterone of adrenal origin may be involved in the onset of cystic ovaries in the sow.     

Role for LDL in estradiol-synthesis capacity of bovine ovarian follicles

The steroidogenic capacity of several tissues has been shown to be regulated by lipoproteins. However, the ability of the various lipoprotein classes to affect steroid production in general and estradiol-synthesis in particular has not been established in bovine ovarian cells. In previous studies, we described alterations in the lipoprotein profiles in follicles of different sizes and corresponding changes in lipoprotein-receptor gene expression. In the present study, the effect of low-density lipoprotein (LDL)-enriched medium on aromatization competence of whole ovarian follicles was determined in vitro. Gene expression of aromatase increased and that of selective-uptake receptors decreased in the presence of LDL. These results suggest a role for LDL availability in folliculogenesis.