Anti-bovine LH Research Articles

CHROMATOGRAPHIC SEPARATION OF THYROID STIMULATING HORMONE FROM LUTEINIZING HORMONE

A new preparative scale procedure is described for the efficient purification of Thyroid stimulating hormone (TSH) from buffalo pituitary glands. The methodology involves the use of hydrophobic interaction chromatography for the separation of TSH from LH which is more abundant than TSH in the pituitary gland. The two hormones were isolated from the pituitary extract by ammonium sulphate precipitation and were then separated on a Phenyl-Sepharose column. The fractions obtained from Phenyl-Sepharose chromatography were assessed using a Uno-Q column on FPLC where enrichment of the TSH in the PS-2 fraction was evident. The final removal of the trace amounts of the LH was done by ion exchange chromatography in tandem on CM-Sephadex and DEAE-Sephacel columns. All the chromatography fractions were assessed in a heterologous direct binding ELISA using bTSH and buLH as the reference hormones and anti ovine TSH beta and anti bovine LH beta as the antisera. The purified fraction (DEAE-0.5 M peak) when checked exhibited no LH immuno reactivity in a western blot against anti bovine LH beta antiserum. Forty two milligrams of a 200 fold purified buTSH per kg wet glands was obtained.

276. The technical and biological validation of an LH assay for use with the Western Grey Kangaroo (Macropus fuliginosis) and Black-flanked Rock Wallaby (Petrogale lateralis lateralis)

Methods for the measurement of marsupial LH invariably rely upon the similarity of the LH molecule between different species and usually use anti-ovine or anti-bovine LH antibody and an ovine or bovine labelled LH preparation. Initial attempts to measure plasma LH in the Western Grey Kangaroo with assays using antibodies to 4 different isoforms of ovine LH raised in 7 different rabbits were unsuccessful. An enzymeimmunoassay (EIA) developed for the Asian elephant (Zoo Biology 23:45–63) was then applied to the Western Grey Kangaroo and the Black-flanked Rock Wallaby. This EIA has an anti-bovine-LH monoclonal antibody (518B7 provided by Dr Jan Roser, University of California, Davis, USA), biotinylated ovine LH label and bovine LH standard (NIADDK-oLH-26 and NIH-bLH-B10, both provided by Dr Janine Brown and Nicole Abbondanza, Smithsonian Institute, Front Royal, Virginia USA). Technical validation showed that serial dilution down to 1:8 of plasma from 7 individuals of each species showed parallelism to the assay standard curve, and control samples (1.24–5.30 ng/mL) had between-assay coefficients of variation <9%. Biological validation was achieved by challenging animals with intramuscular GnRH (Fertagyl®, 2.5 µg/kg) and measuring LH before and 25 min after the injection. Significant increases in plasma concentrations of LH (mean ± sem; all P > 0.0005) were seen after GnRH for both the Western Grey Kangaroo (from 5.0 ± 0.8 ng/mL to 9.4 ± 1.2 ng/mL; n = 19) and the Black-flanked Rock Wallaby (from 6.0 ± 0.7 ng/mL to 10.6 ± 0.6 ng/mL; n = 28). In conclusion, this assay can be successfully used to measure LH in these two species.

Bioactive and immunoreactive concentrations of circulating luteinizing hormone during sexual maturation in the bovine.

The characteristics of circulating LH during sexual maturation in cattle were assessed by examining bioactive and immunoreactive LH concentrations, as well as their ratio (B/I ratio). Male and female intact control (CONT), gonadectomized (GNX; at 241 +/- 3 days of age, Day 0 of the study), and gonadectomized animals administered 17 beta-estradiol (GNXE) were evaluated. Serum samples were collected at 15-min intervals for 24 h at 1, 7, 13, 17, 21, 25, 29, 33, 37, and 43 wk subsequent to Day 0. Bioactive LH was assessed with an in vitro bioassay using mouse testicular interstitial cells. In initial experiments, immunoreactive LH was quantified in RIAs using three different antibodies. The two RIAs employing polyclonal antibodies overestimated low LH concentrations, but the absolute values obtained in each of the three assays were highly correlated. Hence, immunoreactive LH was measured in an RIA using monoclonal anti-bovine LH (bLH) (JR-518B7). No significant changes in the B/I ratios were observed during individual pulses of LH secretion. Accordingly, pools consisting of equal volumes of the serial blood samples collected during the 24-h period for each animal at each stage of maturation (pools) were compared. LH B/I ratios for GNX females increased significantly with time (p less than 0.01) and the B/I ratios for GNX males were significantly higher than for GNX females (p less than 0.05). Concentrations of LH in most of the pools for GNXE and CONT animals were extremely low or nondetectable until the later bleeding periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Development in the cyclic hamster of refractoriness to the superovulatory action of anti-LH serum.

Hamsters injected s.c. on the day of ovulation (Day 1) with 100 microliters equine anti-bovine LH serum ovulated 28 eggs at the end of a 5-day cycle. When a second injection of anti-LH serum was administered 4-93 days later, the animals did not superovulate and had normal 4-day cycles. Injection of 100 microliters normal rabbit serum (NRS) on Day 1 followed 14 days later by anti-LH serum resulted in the ovulation of 32 ova whereas a priming injection of 100 microliters normal horse serum (NHS) followed by anti-LH serum resulted in the ovulation of only 18 ova. When hamsters were injected on Day 1 with anti-LH serum, NHS or NRS and then with anti-LH serum in the 4th cycle, high titres of free antibodies to LH were present on Days 2-4 only in the animals treated with NRS; these hamsters ovulated a mean of 35 ova. These experiments suggest that the hamster rapidly forms antibodies to equine immunoglobulins, thus preventing a second injection of anti-LH serum from inducing superovulation.

Alterations of ovarian steroidogenesis induced by ovulatory delay in immature rats treated with pregnant mare serum gonadotropin.

Immature rats were maintained on a 14L: 1OD schedule (lights on 0600h). Pregnant mare serum gonadotropin (PMSG, 10 IU Primantron, Berlin, Germany) was injected s.c. on Day 28 of age to initiate follicular development for subsequent ovulation of ∿10 ova. Phenobarbital (PB) (6.5 mg/100 g BW) was injected s.c. at 1300 h to induce 1 day of ovulatory delay. Rats treated with 6.5 mg PB on proestrus and 13 mg PB on Day 31 at 1200 h exhibited 2 days of delay; additional injections of 19.5 mg and 26 mg PB were required on the next successive days at 1200 h for 3 and 4 days of ovulatory delay, respectively. During the period of delay, serum concentrations of P remained unchanged; however, serum A and E2 levels decreased significantly. Atresia of the preovulatory follicles was evident in most animals after 4 days of delay. In the remaining experiments, PMSG (from NICHD) was used to initiate follicular development for subsequent ovulation of ∿10 ova. Serum E2 levels on the morning of Day 2 of delay were significantly higher than those levels in rats receiving PMSG (from Primantron) although serum A levels declined and P levels remained unchanged. In vitro ovarian steroidogenesis revealed that the ovaries of rats with a delayed ovulation produced more P and less A than controls in response to 1μg LH (ovine S22) in vitro. Although 1 μg LH in vitro did not induce E2 secretion, ovaries of PB-treated rats produced more E2 than those of nondelayed controls. During the LH/FSH surge (1900 h proestrus), serum P levels increased in groups with 0 and 2 days of ovulatory delay; however, serum A failed to increase after 2 days of delayed ovulation and serum E2 declined. In controls, E2 levels in sera remained unchanged. In vitro ovarian steroidogenesis substantiated the alterations in serum steroids. During 2 days of delayed ovulation, 25 μl equine antibovine LH (anti-LH) administered on Day 30 induced a decline in serum E2 without alteration of serum P and A. Anti-LH (25 μl) given on each day of delay lowered the serum level of A and E2 but not P; 100 μl anti-LH administered on each day of delay lowered serum concentrations of P, A, and E2. Ergocryptine (400 μg CB-154) given on Days 30 and 31 lowered serum prolactin concentrations and partially prevented the decline in serum A levels without alteration of serum P and E2. The results indicate that 1) the PMSG-treated immature rat is a useful model for the study of atresia, 2) ovulatory delay inhibits ovarian A secretion ( and E2, but this depends on the type of PMSG), 3) prolactin may be causally related to the alteration in A synthesis, and 4) preovulatory follicles are sensitive to gonadotropin deprivation during ovulatory delay.

Alteration of the serum follicle-stimulating hormone to luteinizing hormone ratio in the cyclic hamster treated with antiluteinizing hormone: relationship to serum estradiol, free antiluteinizing hormone, and superovulation.

The serum concentrations of unbound LH, FSH, estradiol (E2), and free antibovine LH serum (anti-LH) were determined by RIA in hamsters destined to superovulate (∼32 ova) after a single sc injection of 0.1 ml equine anti-LH at 0900 h on day 1 of the cycle (morning of ovulation). As a control, another group of hamsters was injected with normal horse serum (NHS) at 0900 h on day 1. Animals injected with anti-LH or NHS were decapitated at various times over the next 6 days. Interfering LH antibodies were removed by column chromatography in order to determine the serum concentrations of free FSH and LH by RIA. Serum concentrations of unbound FSH were similar throughout the cycle in animals treated with anti-LH or NHS. On day 2 of the cycle (24 h after anti-LH), free LH levels were less than 6 ng/ml (the lower limit of sensitivity of the RIA on the volume of serum assayed), free anti-LH titers were extremely high, and E2 levels were less than 10 pg/ml. The absence of detectable unbound LH and normal FSH in sera...

Induction of superovulation in the cyclic hamster by a single injection of antiluteinizing hormone serum.

Interruption of pregnancy in the hamster with equine antibovine LH (anti-LH) induces superovulation of about 30 ova at the subsequent estrus. In view of the different hormonal profiles and antral follicular development in pregnant and cyclic hamsters, the present experiments were designed to ascertain the effects of anti-LH on the serum concentrations of steroids, ovarian follicular development, and ovulation in the cyclic hamster. Hamsters injected sc on the morning of ovulation (day 1 of cycle) with 100 μl anti-LH spontaneously ovulated 31.5 eggs at the next ovulation instead of the normal number of 13 ova. The normal 4-day cycle was lengthened to 5 days after anti-LH, but subsequent cycles reverted to 4 days, and the normal number of ova was shed. Anti-LH on day 1 did not affect serum progesterone levels on day 2, but serum estradiol was depressed until day 4, whereas in the normal hamster, estradiol increases on day 3. When anti-LH was given on day 4 (proestrus), the hamsters ovulated the next day an average of 11 ova, but in the subsequent cycle (5–6 days), the mean number of ovulations was 29. Thus, anti-LH induces superovulation when injected in the periovulatory period (days 4 and 1) when serum FSH levels are high. A quantitative study of follicular development after the injection of anti-LH at 0900 h on day 1 revealed that for 12 h afterwards, follicular development was unaffected. However, by day 2, a wave of atresia destroyed the larger preantral follicles. On day 3, the ovary was repopulated with large preantral follicles, and this was followed by a greater than normal number of antral and intermediate sized follicles, thus culminating in superovulation. (Endocrinology108: 1903, 1981)

Antiluteinizing Hormone: Acute Influence on Steroid and Gonadotropin Levels and Follicular Development in the Pregnant Hamster*

Pregnant hamsters were injected at 0900 h on day 4 or 8 (day 1 of pregnancy = sperm-positive) with 0.1 ml of either normal horse serum (NHS) or equine antibovine LH (ALH) and decapitated 1, 3, 6, 12, 18, and 24 h later. Serum was saved for RIA of FSH, PRL, progesterone (P), testosterone (T), and estradiol (E2). Corpora lutea (CL) and nonluteal ovary (NLO) were saved for steroid determinations, and one ovary was saved for histology. Regardless of the day of treatment, ALH induced an immediate decline within 1 h in the serum and luteal E2 concentrations without altering T. At 24 h after ALH, levels of T and P in CL and sera were lower than NHS controls. In NLO, T and E2 declined simultaneously within 1 h after ALH without changing P. Within the next 3–6 h, P in NLO declined to half that of NHS pregnant controls and all antral follicles showed initial signs of atresia. Collectively, these results indicate that, after LH deprivation, the initial hormonal change is a concomitant decrease of E2 in serum, CL, andLO, suggesting that LH is necessary for E2 secretion and maintenance of antral follicles in the pregnant hamster and that decreases of P in serum, CL, and NLO lag behind changes in E2. A dramatic increase in serum FSH-like material accompanied the declines in serum P and E2 after ALH treatment, suggesting that lack of steroid negative feedback at the hypothalamicpituitary axis induces hypersecretion of FSH. A large dose of ALH (0.4 ml) induced a more prompt decline in serum P than 0.1 ml ALH, indicating that a greater degree of LH deprivation may occur sooner with 0.4 ml than with 0.1 ml ALH. Unlike the hysterectomized pseudopregnant (HSP) rat, ALH on day 8 of HSP in the hamster induced a terminal decline in serum P within 24 h. In contrast, the pregnant hamster on day 8 exhibited a more gradual decline in serum P than that observed in the HSP hamster, suggesting that placental factors may be exerting luteotropic actions at this time. The results indicate that luteal aromatizing enzymes appear highly sensitive to LH deprivation since, within 1 h after ALH, luteal E2 concentrations decline without a concomitant decrease in T.

Antiluteinizing Hormone: Chronic Influence on Steroid and Gonadotropin Levels and Superovulation in the Pregnant Hamster*

Pregnant hamsters were injected with either normal horse serum (NHS) or equine antibovine LH (ALH) and autopsied 4 days later (day 1 of pregnancy = sperm positive). Pregnancy was interrupted by a single sc injection of 0.1 ml ALH on days 4, 8, or 10. On days 6 and 12, 0.2 ml ALH was needed to interrupt pregnancy; however, ALH was only partially effective on day 12. After ALH on days 4 or 8 of pregnancy, corpora lutea on days 8 and 12 had regressed and serum progesterone (P) levels were less than 2 ng/ml. However, serum estradiol levels and the number of antral follicles (AF) were not different from NHS controls. There were approximately 11 AF/ovary and 17 AF/ovary in the days 4 and 8 groups, respectively; 4–9 days after ALH, spontaneous superovulation of 23 ± 3 (day 4 group) and 29 ± 4 (day 8 group) ova was observed. In contrast, the rat ovulates only 12.5 ± 0.3 (n = 11) ova after injection of the same ALH (0.2 or 0.5 ml) on day 8 of pregnancy. Serum levels of FSHlike material in ALH-treated hamsters were...

Release of LH and FSH in the normal intact ram by synthetic LH-RF and the effect of pretreatment with testosterone propionate.

were made every 5 min for at least 20 min before the rapid intrajugular injection of LH-RF and then for a further 45 min. Samples were also taken 60 and 90 min after injection and, when 50 \g=m\g LH-RF was administered, the period of frequent sampling was extended. All experiments began at approximately 14.00 hours and at least 3 days were allowed to elapse between experiments. The TP (Koch Light, Colnebrook, Bucks) was administered by intramuscular injection in arachis oil in a 50-mg dose at 09.00 hours and again at 18.00 hours on the first 2 days followed by 100 mg at 09.00 hours on the 3rd day. The res¬ ponse of rams pretreated with TP to a dose of LH-RF was compared with the response to the same dose of LH-RF (7 days earlier) in the same animal pre¬ treated with vehicle only. Plasma LH was assayed by a double antibody method similar in procedure to that of Niswender, Reichert, Midgley & Nalbandov (1969). An equine anti-bovine LH, preabsorbed with bovine TSH (Snook, Saatman & Hansel, 1971) was used at a final dilution of 1 : 200,000. Ovine LH (LER 10 56)

EFFECT OF ANTI-BOVINE LUTEINIZING HORMONE(LH)AND ANTI-RAT PRO-LACTIN(PRL)SERA UPON THE SECRETION OF LH IN INTACT ADULT MALE RATS

Antisera to bovine LH and rat PRL prepared in rabbits were administered s.c. to intact adult male rats daily for 10 days. Control animals received non-immune rabbit serum. Animals were sacrificed by decapitation without anesthesia 24 hrs. after the last injection. Serum and pituitary LH levels were determined by radioimmunoassay. LH data are expressed in terms of NIAMDD Rat LH-RP-1. Testicular and ventral prostate weights were significantly (p < 0.01) reduced in animals who had received anti-bovine LH serum. There were no effects of anti-bovine LH serum upon body, pituitary or adrenal weights. Pituitary LH concentration was significantly higher in anti-bovine LH serum-treated animals than in control rats (31.2±6.5(SD) μg/mg pituitary vs 22.7±6.8 μg/mg, p<0.02). Serum LH levels were higher in animals who had received anti-bovine LH serum than in control rats. Anti-rat PRL serum had no significant effect upon body, pituitary, testicular, ventral prostate or adrenal weights or upon pituitary LH content or concentration. Serum LH concentrations in anti-rat PRL serum-treated animals were higher than in control animals. Inhibition of gonadotropln activity by anti-LH serum resulted in compensatory increase in LH secretion. Data suggest that anti-PRL serum may also interfere with hypothalamic-pituitary-testicular function.

Serum LH levels in prepubertal female pigs chronically treated with synthetic luteinizing hormone-releasing hormone-follicle stimulating hormone-releasing hormone (LH-RH-FSH-RH).

A double—antibody radioimmunoassay utilizing a heterologous system specific for porcine luteinizing hormone (PLH) has been developed with rabbit antibovine LH serum as a first antibody. Porcine LH (LER-786-3) was used for iodination and as a reference standard. Reliable estimates of LH could be obtained in 200 pi of serum from immature pigs at any time. Porcine follicle-stimulating hormone (FSH), porcine thyroid- stimulating hormone, porcine adrenocorticotropic hormone or serum components per se did not affect measurements of LH. Ten prepubertal female pigs (mean wt 12.8 kg) were assigned randomly in equal numbers to a treated or control group. Treatment consisted of 16 im injections of 25 ng of pure synthetic LH and FSH releasing hormone (LH-RH/FSH-RH) in one ml of acidified saline or acidified saline only at 6–hr intervals over a period of 96 hr for treated and control animals respectively. Blood samples were collected via indwelling jugular catheters, at various times following each injection. Serum LH...

Survival and gonadotrophin responsiveness of luteal cells in vitro.

ABSTRACT Simplified methods for incubating luteal tissues have been developed. Progesterone biosynthesis in washed, minced bovine luteal tissue is stimulated by added bovine LH. The response is linear in the range 2–200 ng added LH per ml of medium. The use of covariance analysis to correct for differences in time elapsing between mincing the tissue and the beginning of incubation reduces the standard error of the mean and results in marked improvement in the indices of precision. Progesterone biosynthesis in washed, minced bovine luteal tissues appears specific for LH; no other pituitary hormones give a response, and the response is completely negated by adding anti-bovine LH serum to the incubation medium. Prior treatment of cattle from which the incubated corpora lutea are obtained with various levels of human chorionic gonadotrophin, results in greatly increased luteal tissue weights. However, this tissue has a markedly reduced sensitivity to LH added in vitro. No conclusive evidence was found for a feedback inhibition of progesterone on its own synthesis in this system. The incubation system developed is sensitive enough to serve as a bioassay for LH in biological tissues and fluids. Preliminary data suggest that a simplified protein binding assay can be successfully used to measure the progestins synthesized by minced, washed bovine luteal tissue in response to added bovine LH.

Termination of Pregnancy in the Rat with Rabbit Antibovine LH Serum

Termination of Pregnancy in the Rat with Rabbit Antibovine LH Serum

The effect of antisera to bovine LH in hysterectomized and intact heifers.

Injection of antiserum to bovine LH into hysterectomized heifers caused significant decreases in corpus luteum weight, total progesterone, total progestins and 20�-hydroxypregn4-en-3-one (204-01) concentration but had no effect on progesterone concentration. Gonadotropin secreting cells in the anterior pituitaries of these heifers were markedly degranulated and pituitary LH contents lowered. Thyroid epithelial cell heights were lowered suggesting that the antiserum also contained anti-TSH. The administration of antibovine LH to cyclic heifers from Day 2 to Day 6 of the cycle caused a significant decrease in corpus luteum weight and total progesterone content, but only a slight decrease in progesterone concentration. Histological signs of regression were clearly evident in the corpora lutea of the hysterectomized anti-LH treated heifers, but little difference could be noted between the corpora lutea of control and treated cyclic animals. Injection of 50 ml of antibovine LH daily from Day 2 to Day 6 of the cycle increased the interval between corpus luteum removal at Day 11 and return to estrus. Injections of 50 ml of antibovine LH daily from Day 12 to Day 16 and from Day 16 to Day 20 of the cycle increased the lengths of the treatment cycles to 28.5 and 36.7 days, respectively. The first post-treatment cycle in both groups was significantly shortened. The increase in cycle length was primarily due to a failure of follicles to develop to mature size, rather than to prolongation of the life span of the corpora lutea. The results of these experiments indicated that LH is required for follicular development and maturation and for the induction of estrus, as well as for corpus luteum maintenance.

Maintenance of Luteal Function in the Cow

A series of experiments conducted over a period of several years has provided the following evidence that LH plays a major role in stimulating corpus luteum growth and progesterone synthesis in cattle:Purified bovine LH and other LH-containing preparations overcome the inhibitory effects of concurrently injected oxytocin on luteal tissue weight, progesterone content, and concentration; whereas, other pituitary hormones, including prolactin, FSH, and growth hormone are incapable of overcoming the inhibitory effects of oxytocin.Single injections of purified bovine LH in Freund's adjuvant given at mid-cycle significantly prolong the functional life span of the corpus luteum and lengthen the estrous cycle.LH-containing preparations and crude bovine anterior pituitary extracts significantly increase corpus luteum weights and progesterone contents in hysterectomized heifers. The administration of equine anti-bovine LH serum causes partial regression of corpora lutea in hysterectomized heifers. Incubation of LH-containing preparations with urea abolishes their luteotropic properties.Progesterone synthesis in vitro by bovine luteal tissue slices is stimulated by purified bovine LH. The reaction is highly sensitive, specific, and is inhibited by addition of equine anti-bovine LH serum.