卵泡抑制素主动和被动免疫及基因免疫研究进展

抑制素是由动物卵巢颗粒细胞或睾丸间质细胞分泌的糖蛋白激素 ,对垂体FSH的分泌具有抑制作用。用抑制素或其片断主动或被动免疫可以提高家畜的排卵率和产仔数 ;用抑制素基因免疫可望得到类似效果。本文通过分析抑制素的理化特性、生物学作用及作用机制、主动和被动免疫对家畜繁殖力的影响 ,阐述了抑制素基因免疫的原理与方法及应用前景     

Circulating concentrations of immunoreactive inhibin and FSH during the estrous cycle of mares

A heterologous radioimmunoassay developed to measure inhibin in rat plasma was validated and used to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to follicle stimulating hormone (FSH) concentrations during the estrous cycle of mares. The primary antiserum used in the assay was developed against a synthetic porcine inhibin -subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin -subunit standard curve and porcine follicular fluid dose-response curve. Twelve mares were bled once daily beginning when diameter of the largest follicle reached ≤25 mm and continuing until 3 days after the end of an interovulatory interval (ovulation=Day 0). Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26). There was an effect of day for concentrations of ir-inhibin (P<0.001) and FSH (P<0.006). Significant mean changes were as follows: 1) ir-inhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) ir-inhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean concentrations of ir-inhibin and FSH were negatively correlated (r=-0.548; P<0.002). In conclusion, mean peripheral concentrations of ir-inhibin and FSH were inversely related during the estrous cycle of mares.     

Follicular and hormonal dynamics during the estrous cycle in goats

Transrectal ultrasonography of ovaries was performed daily in 6 goats for 3 consecutive estrous cycles. Blood samples collected daily were measured for concentrations of FSH, inhibin A, and estradiol-17beta. Follicular and hormonal data were analyzed for associations between the follicular waves and hormonal concentrations. During the interovulatory intervals, follicular growth and regression occurred in a wave like pattern (2-5 waves), and the predominant patterns were three and four follicular waves. In addition, there was no significant difference among the diameters of dominant follicles during the growth phase of the follicular waves. The number of 3 mm follicles peaked on days 0, 7, and 11 in interovulatory intervals that had three follicular waves and on days -1, 5, 11, and 15 in those that had four follicular waves. Plasma concentrations of FSH increased around the day of follicular wave emergence and declined with the growth of follicles. Circulating FSH increased again concomitant with regression of dominant follicles in the anovulatory wave, whereas FSH levels remained low in the ovulatory wave. Inhibin A was negatively correlated with FSH, while it was positively correlated with estradiol-17beta, suggesting that inhibin A is a product of healthy growing follicles and that it contributes to the suppression of FSH secretion. In conclusion, the growth of ovarian follicles in goats exhibits a wave-like pattern, and follicular dominance is less apparent in goats. Moreover, inhibin A may be a key hormone for regulation of the follicular wave through suppression of FSH secretion in goats.     

Effect of active immunization of pony mares against recombinant porcine inhibin alpha subunit on ovarian follicular development and plasma steroids and gonadotropins

Two pony mares were immunized against recombinant porcine inhibin alpha subunit three times with 39 day intervals. Clinical findings and endocrinological changes before immunization were taken as the control. The first significant rise in the anti-inhibin titre (P<0.05) in the circulation was found 27 days after the first injection. Maximum binding activity was reached by the 12th day after the second booster dose. The number of small, medium and large sized follicles had increased significantly compared to before immunization (11.75 +/- 4.30, 2.75 +/- 0.69 and 2.51 +/- 0.63 vs 6.50 +/- 1.43, 1.83 +/- 0.44 and 1.33 +/- 0.38, respectively), but the ovulation rate remained unchanged after immunization. The average plasma concentration of FSH and estradiol-17beta during the estrous cycle increased significantly (P<0.05) after immunization. These results suggest that immunization against inhibin is a useful tool to increase the number of ovarian follicles during the estrous cycle of pony mares. Moreover, the present study supported the concept that inhibin plays a major role in the control of follicular growth through its inhibitory effect on FSH secretion synergistically with steroid hormones.     

Passive immunization of cyclic mares against androgen: gonadotropin and progesterone concentrations and estrous characteristics

Antiserum generated in a horse against testosterone conjugated to bovine serum albumin (BSA) was administered to six lighthorse mares (androgen-immunized mares) 1 to 3 d before a prostaglandin-induced estrus and twice again at 2-d intervals. Six control mares were administered antiserum generated against BSA on the same schedule. Relative to testosterone, cross-reactivities of other steroids with the testosterone antiserum were (%): dihydrotestosterone, 52; 5 alpha-androstane-3 alpha,17 beta-diol, 8.6; androst-4-ene-3,17-dione, 1.2; and all others tested less than .1. Tritiated testosterone binding in plasma increased (P less than .01) in androgen-immunized mares within 1 h and remained elevated (P less than .01) relative to controls for greater than 21 d. There was no effect (P greater than .10) of passive immunization against androgen on interval to estrus after prostaglandin injection, duration of estrus, ovarian volume, number of palpable follicles or follicular volume during estrus. In contrast, concentrations of luteinizing hormone (LH) were higher (P less than .05) in androgen-immunized mares than in control mares during estrus and early diestrus. Concentrations of follicle stimulating hormone (FSH) and progesterone at those times were not affected (P greater than .10). From these data, we conclude that androgens in the mare during estrus may be involved with the regulation of LH secretion. In contrast, no involvement with FSH secretion was apparent under these short-term conditions.     

牦牛发情周期中卵巢卵泡发育状况的组织学观察

运用组织学技术对36头处于发情周期的健康成年母牦牛卵巢卵泡的结构与发育状况进行了观察。结果表明，牦牛发情周期中卵巢卵泡发育的组织结构与其他牛基本相似。每对卵巢中原始卵泡数和生长卵泡数在发情前期、发情期、发情后期和发情间期之间差异不显著（P〉0．05）；发情前期的囊状卵泡数与发情期、发情后期和发情间期之间差异不显著（P〉0．05），发情期和发情后期均与发情间期差异显著（P〈0．05）。闭锁生长卵泡数在4个时期之间差异不显著（P〉0．05）；发情期的闭锁囊状卵泡数与其他3期之间差异极显著（P〈0．01），发情前期和发情间期均与发情后期差异显著（P〈0．05）。各级卵泡的闭锁形式各有特点。在生长卵泡、囊状卵泡及相应闭锁卵泡的卵泡膜中均见到了胶原纤维和网状纤维。     

Pituitary and testicular responses of beef bulls to active immunization against inhibin alpha

Prepubertal crossbred beef bulls served as controls or were actively immunized against the N-terminal, 30-amino acid synthetic fragment of porcine inhibin alpha, pI alpha (1-30). Antibody titers were detected in sera (greater than 40% B/BO in sera diluted 1,000-fold) but not in rete testis fluid of 390-d-old bulls. Serum FSH and inhibin remained static during a 5-h intensive bleed; inhibin was not acutely affected by a 15-fold LH rise and a threefold FSH rise induced by exogenous GnRH. Serum FSH, but not LH or testosterone, was consistently elevated (P less than .05) in immunized bulls compared with control bulls. Neither pituitary weight, pituitary gonadotropin content nor pituitary FSH/LH ratios were affected (P greater than .10) by pI alpha(1-30) active immunization. Testicular sperm density was greater (60 x 10(6) vs 45 x 10(6) sperm/g testis; P less than .10) in immunized bulls, but testes weight, epididymides weight and total daily sperm production remained unchanged. These results suggest that inhibin is important for regulation of FSH secretion and testicular function. Immunization with suitable inhibin vaccines may improve bull fertility.     

Effects of different five-day progesterone-based synchronization protocols on the estrous response and follicular/luteal dynamics in dairy cows

This study compared the responses shown by lactating dairy cows to four different P4-based protocols for AI at estrus. Cows with no estrous signs 96 h after progesterone intravaginal device (PRID) removal were subjected to fixed-time AI (FTAI), and their data were also included in the study. In Experiment I, follicular/luteal and endometrial dynamics were assessed every 12 h from the beginning of treatment until AI. The estrous response was examined in Experiment II, and fertility was assessed in both experiments. The protocols consisted of a PRID fitted for five days, along with the administration of different combinations of gonadotropin releasing hormone (GnRH), equine chorionic gonadotropin and a single or double dose (24 h apart) of prostaglandin F_2α. In Experiment I (40 cows), animals receiving GnRH at the start of treatment showed a significantly higher ovulation rate during the PRID insertion period while estrus was delayed. In Experiment II (351 cows), according to the odds ratios, cows showing luteal activity at the time of treatment were less likely to show estrus than cows with no signs of luteal activity. Treatment affected the estrous response and the interval from PRID removal to estrus but did not affect conception rates 28–34 days post AI. Primiparous cows displayed a better estrous response than multiparous cows. Our findings reveal acceptable results of 5-day P4-based protocols for AI at estrus in high-producing dairy cows. Time from treatment to estrus emerged as a good guide for FTAI after a 5-day P4-based synchronization protocol.     

Plasma concentrations of luteinizing hormone and adrenocorticotropic hormone in blood collected during the luteal and follicular phases of the estrous cycle in cows.

Luteinizing hormone (LH) and ACTH concentrations were measured in plasma from 7 cows to determine whether ACTH secretion changes with the phase of the estrous cycle, and to determine whether any ACTH peaks are associated with LH peaks. Blood was collected every 5 minutes for 190 minutes during the luteal and follicular phases of the estrous cycle. Radioimmunoassays were used to measure ACTH and LH in plasma. Mean concentration of ACTH in all cows did not differ significantly between luteal (35.1 +/- 8.0 pg/ml) and follicular (37.5 +/- 9.4 pg/ml) phases of the estrous cycle. Mean concentration of luteal-phase LH of all cows (2.0 +/- 1.1 ng/ml) was significantly (P less than 0.01) lower than mean concentration of follicular-phase LH (5.4 +/- 1.6 ng/ml). Frequency of peaks in ACTH concentration was low during the sampling period. Mean number of luteal-phase ACTH peaks (0.29 +/- 0.49) was not significantly different from that of follicular-phase samples (0.43 +/- 0.530). Unlike ACTH, mean frequency of LH peaks was significantly (P less than 0.05) higher in plasma from cows in the follicular phase of the estrous cycle (2.9 +/- 0.7), compared with that from cows in the luteal phase (0.29 +/- 0.49).     

Production of estradiol by each ovary during the estrous cycle of cows

The objective of our experiment was to examine changes in serum concentrations of estradiol in each utero-ovarian vein before, during and after gonadotropin surges. Four cows were given prostaglandin F2 alpha (PGF2 alpha) during diestrus and three cows were allowed to cycle spontaneously. All cows had a cannula in each utero-ovarian vein and in one jugular vein. Most cows had two transient rises in estradiol, primarily coming from a single ovary, preceding and after luteinizing hormone (LH) surges. The first rise in estradiol began after luteal regression and was sustained from 48 h before a pre-ovulatory LH surge to the end of the LH surge. The second rise in estradiol was sustained from 72 to 168 h after the end of an LH surge. To determine how rapidly asymmetrical production of estradiol began during luteolysis, several cows were injected with PGF2 alpha during the luteal phase. Blood samples were taken from a jugular and both utero-ovarian veins at hourly intervals before and after PGF2 alpha. Asymmetrical production of estradiol began within 3 h after an injection of PGF2 alpha. We concluded: (1) that a single ovary was responsible for the sustained increases in concentration of estradiol that occur during proestrus to estrus and early diestrus in cows and (2) that cows may have at least one follicle capable of producing estradiol during most days of an estrous cycle, thus little delay in selection of which follicle eventually ovulates occurs after luteal regression.     

Changes in hormonal profiles during the estrous cycle in old lactating beef cows

Patterns of concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (P4) and estradiol-17 beta (E2) during an estrous cycle were compared between 15 lactating beef cows 5 to 7 years of age (young) and 15 cows greater than or equal to 12 years of age (old). Length of estrous cycle did not differ between young and old cows (P = .06). No differences due to age were found for LH. Patterns of concentrations of P4 during the first 15 days of the cycle, of FSH during days 6 through 12 and of E2 during the follicular phase differed with age (P less than .05). An earlier (P less than .025) midcycle elevation of FSH was associated with an earlier rise and greater concentration of E2 (P less than .05) during the follicular phase in old than in young cows. Differences in FSH and P4, although subtle, were consistent with an earlier or more advanced follicular development in old cows, leading to greater secretion of E2 from the preovulatory follicle.     

Submicroscopic changes in the glandular epithelium of the endometrium in cows during the estrous cycle

During the sexual cycle the structural changes in the cells of the glandular epithelium in the endometrium of a cow are more pronounced than those in the luminal epithelium. Microvilli appear on cell surface during pro-oestrus; in later stages the microvilli disappear and the apical parts penetrate into the lumen. The relative volume of mitochondria increases from pro-oestrus to culminate in oestrus and metoestrus and to drop in dioestrus. The values of the volume and surface area of granular endoplasmic reticulum are also at their highest level in oestrus and metoestrus. The lowest volume and surface area of smooth membranes are recorded in dioestrus, then it markedly increases to culminate in metoestrus. The incidence of lysosomes is variable and their volume culminates in metoestrus. The proliferation of the glandular epithelium has the highest intensity in oestrus and reaches its peak values in metoestrus when the apical parts of cytoplasm contain the largest amounts of smooth-walled vesicles and vacuoles, partly also secretory granules, and when the signs of secretion are observed.     

Ovarian follicular dynamics and hormones throughout the estrous cycle in Thai native (Bos indicus) heifers

Relatively few studies have been reported regarding the reproductive physiology of female Thai native cattle. Therefore, the objective of the present study was to evaluate the follicular dynamics and concentrations of follicle stimulating hormone (FSH), estradiol (E2) and progesterone (P4) during the estrous cycle in Thai native heifers (TNH) and to compare obtained results with those of European and Indian cattle breeds previously reported. For the detection of estrus, ovaries of all 20 heifers were examined twice daily (12 h intervals) by ultrasonography for three consecutive estrous cycles. From data of 60 estrous cycles (n = 60 estrous cycles from 20 heifers), it was found that 14 (70%) and 6 heifers (30%) had two (42 estrous cycles collected from 14 heifers) and three follicular waves (18 estrous cycles collected from 6 heifers), respectively. The days when estrus was detected, interovulatory intervals, life‐spans of corpus lutea (CL), and days for growing and regression of CLs were shorter in the two follicular waves than those in the three follicular waves (P < 0.05). In both two and thre follicular waves, larger maximum diameters and higher growth rates of the dominant follicle (DF) in an ovulatory wave were observed than those of the preceding waves without ovulation (P < 0.05). There was a progressive increase in follicular size and FSH and E2 production during follicular growth in each follicular wave. In addition, the FSH and E2 peak concentrations during the ovulatory wave were higher than those of the anovulation waves (P < 0.05). Moreover, although the ovarian follicular dynamic patterns in Thai native heifers were similar to those previously reported for European and Indian cattle breeds, the diameter of the largest preovulatory follicle (OF), subordinate follicles (SF) and CLs were smaller than those in European and Indian cattle breeds. In conclusion, when compared with European and some breeds of Indian cattle, the length of interovulatory intervals was shorter, and the sizes of dominant SF and CLs were smaller in Thai native heifers.     

Effects of active immunization against gonadotropin releasing hormone on gonadotropin secretion after ovariectomy and testosterone propionate administration to mares

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) conjugated to bovine serum albumin (BSA) to study the involvement of GnRH in luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion following ovariectomy (OVX) and after administration of testosterone propionate (TP). Five mares immunized against BSA served as controls. Immunizations were started on November 1, and OVX was performed in June (d 1). All mares were treated with TP from d 50 to 59 after OVX. On the day of OVX, concentrations of LH were lower (P less than .05) in GnRH-immunized mares than in BSA-immunized mares and were generally nondetectable; FSH concentrations were reduced (P less than .05) by 50% in GnRH-immunized mares relative to BSA-immunized mares. In contrast to BSA-immunized mares, plasma concentrations of LH or FSH did not increase after OVX in GnRH-immunized mares. The LH response to GnRH analog (less than .1% cross-reactive with GnRH antibodies) on d 50 was reduced (P less than .05) by 97% in GnRH-immunized mares relative to BSA-immunized mares, whereas the FSH response was similar for both groups. Treatment with TP for 10 d reduced (P less than .01) the LH response and increased (P less than .01) the FSH response to GnRH analog in BSA-immunized mares, but it had no effect (P greater than .1) on the response of either gonadotropin in GnRH-immunized mares.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of an orally active progestin on follicular dynamics in cycling and anestrous postpartum beef cows

Although treatment of cycling cows with low concentrations of melengesterol acetate (MGA) results in formation of persistent follicles, in the absence of corpora lutea, it is not known whether persistent follicles form in anestrous cows in response to a similar treatment. The objective of this experiment was to determine the effect of long-term MGA treatment (14 d) on follicular dynamics and the secretion of estradiol in anestrous postpartum beef cows. Treatment groups (replicated over 2 yr) included the following: anestrous control (AC; n = 11), anestrous MGA (AM; n = 16), and cycling MGA (CM; positive control; n = 16). Angus-crossbred cows were assigned to treatment by age, cow body condition, and days postpartum. Cows were fed carrier (AC group) or 0.5 mg MGA x animal(-1) x d(-1) (AM and CM groups) for 14 d beginning approximately 38 d postpartum. Cows allotted to the CM group were injected with PGF2alpha, on the first day of MGA treatment to induce luteolysis. The preceding treatment (CM) results in formation of persistent follicles and secretion of elevated concentrations of estradiol. Ovaries of each cow were examined daily by transrectal ultrasonography beginning 5 to 7 d preceding the initiation of feeding MGA or carrier and continued until ovulation or 7 d following MGA feeding. There was no difference among groups in the stage of follicular wave or diameter of the largest follicle at the start of carrier or MGA feeding. The length of the follicular wave present at the start of MGA feeding was greater (P < 0.01) for cows in the CM (14.5 d, yr 1; 18.3 d, yr 2) group compared to the AM (9.4 d, yr 1; 7.9 d, yr 2) or AC (9.7 d, yr 1; 10.7 d, yr 2) groups. Maximum follicular diameter over both years was greater (P < 0.01) for the CM (20.6 mm) group than the AM (15.1 mm) or AC (16.4 mm) groups. Circulating concentrations of estradiol were also increased (P < 0.05) in the CM group compared to the AM or AC groups. However, MGA appeared to have no effect (P > 0.05) on the number of follicles recruited, growth rate of the dominant follicle during the first 6 d oftreatment, or growth rate to the maximum follicular diameter. In summary, MGA treatment did not increase the duration ot the follicular wave, maximum follicular diameter, or secretion of estradiol in anestrous postpartum cows, nor did MGA affect the number of follicles recruited or growth rate of dominant follicles in cycling or anestrous animals.     

The effect of GnRH or oestradiol injected at pro-oestrus on luteal function and follicular dynamics of the subsequent oestrous cycle in non-lactating cycling Holstein cows

Oestrous synchronization involves synchronization of ovarian follicular turnover, new wave emergence, and finally induction of ovulation. The final step can be synchronized by the parenteral administration of either GnRH or oestradiol benzoate. This study investigated corpus luteum and follicular emergence after ovulation had been induced by the administration of either GnRH or oestradiol benzoate. The injection of oestradiol benzoate may have delayed the emergence of the first follicular wave subsequent to the induced ovulation; administration of oestradiol benzoate or GnRH lowered the progesterone rise so that the maximum dioestrous concentration of progesterone on Day 9 was lower when cows were treated during pro-oestrus compared to the spontaneously ovulating controls. One implication of findings from the present study is that induction of ovulation with either oestradiol benzoate or GnRH, administered 24 or 36 h after withdrawal of the CIDR device, respectively, may lower fertility. Future studies must identify the timing of administration relative to the time of CIDR device withdrawal and the optimum concentration of oestradiol benzoate or GnRH that would not have untoward effects on the development of the corpus lutea, particularly within the first week of dioestrus.     

Comparison of leptin levels in serum and follicular fluid during the oestrous cycle in cows

Leptin is mainly synthesised in white adipose tissue. Besides its effects on body weight and metabolic homeostasis, leptin also has effects on puberty, sexual maturation and reproduction. In this study the relationship between leptin, IGF-1, oestradiol (E2) and progesterone levels were investigated in serum and follicular fluid from cows. This study included 72 healthy, Brown Swiss cows aged 4-5 years. Samples from the jugular vein and follicular fluids were collected. Phases of the oestrus cycle of cows were classified according to their serum progesterone levels (< 3.18 nmol/l, follicular phase and the others as luteal phase). Follicles were grouped as large (> or = 8 mm) or small (< 8 mm). Leptin, IGF-1, oestradiol and progesterone levels were measured from serum and follicular fluid. Leptin concentrations were found to be significantly higher in luteal-phase follicular fluid of small follicles (P < 0.05). These were classified as atretic follicles. There was a positive correlation between serum and follicular fluid leptin levels in the luteal phase. Serum leptin was found to have a positive correlation with follicular fluid progesterone level (P = 0.01) in the preovulatory follicles. The present study shows that there is a relationship between the concentration of leptin in follicular fluid and atresia in small follicles.     

The effect of active immunization of ewes against androstenedione on lambing rate and production

Active immunization of sheep of the Dala breed against androstenedione caused a mean increase in number of lambs born (live-born and stillborn) of 0.29 (p less than 0.01). In yearlings the difference was 0.43 lambs (p less than 0.01) and in older ewes 0.23 lambs (p less than 0.1). Five of the older immunized ewes gave birth to quadruplets while none in the control group gave birth to more than 3 lambs. In older ewes there was an increase in the number of stillborn lambs. In young immunized ewes the mean ewe production, registered as total live weight of lambs in the autumn, was 11.3 kg higher than in control animals. This difference was however not significant (p less than 0.1).