ABSTRACT

Two experiments were conducted on White Leghorn hens to investigate the effect of different concentrations of LH on the frequency of induction of ovulation in vitro of ovarian follicles collected 18–20 h before the expected time of ovulation from birds of three different ages (7, 18 and 30 months) or of the same age but laying short (three to four) or long (more than seven) sequences of eggs. The ability of LH to induce ovulation was directly related to the age of the donors of the follicles. Follicles derived from hens laying short sequences of eggs were less responsive to the ovulation-inducing effects of LH than were follicles from birds laying long sequences. These observations suggest that the sensitivity of ovarian follicles to the ovulation-inducing effects of LH declines with age and is greater in hens laying long sequences of eggs than those laying short sequences.

J. Endocr. (1985) 106, 67–69

SUMMARY

Changes in four hydrolytic enzymes, namely acid phosphatase, alkaline phosphatase, arylsulphatase A and B, of the cervix of the rat and hamster have been studied during the 4-day oestrous cycle. All four enzymes showed maximal activity on the day of oestrus and least activity on day 2 of dioestrus. All the enzymes showed significant reduction of activity after ovariectomy, arylsulphatase A and B showing the earliest changes in specific activity.

A single subcutaneous injection of 0·02 μg oestradiol-17β/rat increased the specific activity of arylsulphatase A and B from the low ovariectomized level to that observed in control oestrous animals within 18 and 6 h respectively. A higher concentration of oestradiol-17β (2·0 μg) had an inhibitory effect. Progesterone was without effect on arylsulphatase B activity, but when given (2·0 mg) with 0·02 μg oestradiol-17β, it inhibited the response to oestrogen.

Cycloheximide prevented the rise in arylsulphatase B activity occurring after oestrogen injection, suggesting a regulation of cervical arylsulphatase B at the level of protein biosynthesis. These results suggest that arylsulphatase B activity may be induced by oestrogen in the cervix of the rat.

Abstract

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)–Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20–30 μg of the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of ¹²⁵I-LH/CG-R added and (2) analysing sera for any change in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2·5- to 6·0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (∼40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED₅₀) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in anti-body titre (ED₅₀ of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity of antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) ¹²⁵I-hCG binding to crude sheep luteal membrane (EC₅₀ of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence of antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 430 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 μg). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with holo LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Journal of Endocrinology (1996) 150, 431–443

ABSTRACT

The role of FSH and diurnal testosterone rhythms in specific germ cell transformations during spermatogenesis were investigated using DNA flow cytometry and morphometry of the seminiferous epithelium of the adult male bonnet monkey (Macaca radiata), the endogenous hormone levels of which were altered by two different protocols. (1) Active immunization of five monkeys for 290 days using ovine FSH adsorbed on Alhydrogel resulted in the neutralization of endogenous FSH, leaving the LH and diurnal testosterone rhythms normal. (2) Desensitization of the pituitary gonadotrophs of ten monkeys by chronically infusing gonadotrophin-releasing hormone analogue, buserelin (50 μg/day release rate), via an Alzet pump implant (s.c.) led to a 60–80% reduction in LH and FSH as well as total abolition of testosterone rhythms. The basal testosterone level (3·3±2·0 μg/l), however, was maintained in this group by way of an s.c. testosterone silicone elastomer implant. Both of the treatments caused significant (P <0·01) nearly identical reduction in testicular biopsy scores, mitotic indices and daily sperm production rates compared with respective controls. The germ cell DNA flow cytometric profiles of the two treatment groups, however, were fundamentally different from each other. The pituitary-desensitized group exhibited a significant (P <0·001) increase in 2C (spermatogonial) and decrease in 1C (round spermatid) populations while S-phase (preleptotene spermatocytes) and 4C (primary spermatocytes) populations were normal, indicating an arrest in meiosis caused presumably by the lack of increment in nocturnal serum testosterone.

In contrast, in the FSH-immunized group, at day 80 when the FSH deprivation was total, the primary block appeared to be at the conversion of spermatogonia (2C) to cells in S-phase and primary spermatocytes (4C reduced by >90%). In addition, at this time, although the round spermatid (1C) population was reduced by 65% (P <0·01) the elongate spermatid (HC) population showed an increase of 52% (P <0·05). This, taken together with the fact that sperm output in the ejaculate is reduced by 80%, suggests a blockade in spermiogenesis and spermiation. Administration of booster injections of oFSH at time-points at which the antibody titre was markedly low (at days 84 and 180) resulted in a transient resurgence in spermatogenesis (at day 180 and 228), and this again was blocked by day 290 when the FSH antibody titre increased. Although in the two treatment groups overall spermatogenesis, as indicated by 1C:2C ratios, was significantly (P <0·01) reduced, the mechanisms by which this was brought about appeared to be different, suggesting that lack of FSH and testosterone (in particular the nocturnal testosterone surge) affect germ cell transformation at different foci.

Journal of Endocrinology (1993) 137, 485–495

Abstract

Sexually mature male rabbits actively immunized against highly purified ovine LH (oLH) were used as a model system to study the effects of endogenous LH deprivation (and therefore testosterone) on spermatogenesis as well as pituitary FSH secretion. Immunization against oLH generated antibody titres capable of cross-reacting and neutralizing rabbit LH and this resulted in a significant reduction (P<0·01) in serum testosterone levels by 2–4 weeks of immunization. A significant increase in circulating FSH concentration (from a basal level of ∼ 1 ng to 60–100 ng/ml; P<0·01) was observed within 4–6 weeks of immunization, perhaps a consequence of the negative feedback effect of the lack of testosterone. The effect of LH deprivation on spermatogenesis assessed by DNA flow cytometry and histological analyses of testicular biopsy tissue revealed that lack of testosterone primarily results in a rapid reduction and complete absence of round (1C) and elongated (HC) spermatids. The immediate effect of LH/testosterone deprivation thus appears to be at the step of meiotic transformation of primary spermatocytes (4C) to 1C. A significant reduction (>80%; P<0·01) in the 4C population and a relative accumulation (>90%; P<0·01) in spermatogonia (2C) was also observed, suggesting a need for testosterone during the transformation of 2C to 4C. In all but one of the rabbits, both qualitative and quantitative recovery in spermatogenesis occurred during the recovery phase, even at a time when only a marginal increase in serum testosterone (compared with the preimmunization) levels was observed as a result of a rapid decline in the cross-reactive antibody titres. These results clearly show that LH/testosterone deprivation in addition to primarily affecting the meiotic step also regulates the conversion of 2C to 4C during spermatogenesis.

Journal of Endocrinology (1995) 147, 111–120

SUMMARY

The effect of a single injection of luteinizing hormone (LH) antiserum on ovarian progesterone and 20α-dihydroprogesterone in day-8 and day-15 pregnant rats was studied. Within 24 h of an injection of LH antiserum, progesterone secretion was reduced by 80% in day-8 and by 25% in day-15 pregnant rats. The 20α-dihydroprogesterone levels after antiserum treatment were markedly increased in rats which were 8 days pregnant but reduced in rats which were 15 days pregnant. The free cholesterol content of the ovary did not change after antiserum injection but the cholesteryl ester content markedly increased. It is thus apparent that neutralization of endogenous LH resulted in a significant reduction in the progesterone secretion of the corpus luteum of the pregnant rat. The significance of these results is discussed.

SUMMARY

The effect of antiserum to luteinizing hormone (LH) on progesterone and 20α-dihydroprogesterone output and on the enzymes regulating luteal cholesteryl ester turnover was measured in pseudopregnant rats to provide information on the role of LH in regulating luteal function. Progesterone synthesis was reduced when antiserum was added directly to an incubation system of luteinized ovarian slices from animals which had received intravenous saline or 10 μg LH. Steroidogenesis was stimulated in vitro when LH was previously given in vivo, and also on incubating luteinized ovaries from animals treated with antiserum. Treatment in vivo with antiserum alone, however, increased progesterone and 20α-dihydroprogesterone synthesis in vitro but this appeared to be an effect of incubation since in a separate experiment peripheral serum levels of both progesterone and 20α-dihydroprogesterone were reduced after antiserum treatment. Ovarian levels of cholesteryl ester and cholesterol were increased after antiserum treatment in vivo. The level of ovarian cholesteryl esterase was reduced to only 10% of the control level 24 h after antiserum treatment and in this period the level of cholesteryl ester synthetase increased 1·5 times. From these results LH appears to play a direct role in regulating the activity of enzymes controlling cholesteryl ester turnover and thereby regulates the availability of cholesterol for conversion to steroids.

SUMMARY

The follicle-stimulating hormone (FSH) inhibitor in monkey urine was purified by selective extraction of the crude extract with acetate buffer, ammonium sulphate precipitation and DEAE-cellulose chromatography. The purified inhibitor was free of luteinizing hormone activity. It behaved as an apparently homogeneous protein. The inhibitor contained about 20% carbohydrate (hexoses, hexosamines, fucose and sialic acid). Thin-layer gel filtration indicated a molecular weight of about 65,000. The inhibitor was labile to heat treatment, exposure to extremes of pH and denaturing agents. The inhibitor effectively neutralized the biological activity of FSH preparations from human, monkey, horse, pig, sheep and rat pituitary glands, pregnant mare serum gonadotrophin and human pituitary urinary gonadotrophin.

Abstract

While the endocrine role of oestrogen is well established, its function in follicular maturation as an autocrine or paracrine regulator is less well understood. This study was designed to delineate the requirement of oestrogen for follicular development in immature rats. Exogenous gonadotrophin (25 IU pregnant mare serum gonadotrophin (PMSG) per rat) was administered to 21- to 23-day old female rats to induce follicular growth and development. In the experimental animals, synthesis of oestrogen was blocked by implanting an Alzet pump containing the aromatase inhibitor (AI) CGS 16949A (fadrozole hydrochloride; 50 μg/rat per day). The treatment resulted in blockade of the PMSG induced increase in both serum and intrafollicular oestrogen (>95%), thus leading to an inhibition in uterine weight increment. Compared with the controls, ovarian weight increased markedly in both the PMSG (295%)- and PMSG+AI (216%)-primed animals.

There was no significant difference in either the proliferative capabilities of the ovarian granulosa cells or their responsiveness to human chorionic gonadotrophin (hCG; 200 pg/ml) and ovine FSH (20 ng/ml) between the PMSG- and PMSG+AI-treated groups. Histological examination of the ovary, however, indicated a decrease in the number of healthy antral follicles in the AI-treated group compared with the PMSG-primed animals but both the groups showed a percentage increase over the controls (PMSG, 225; PMSG+AI, 158). The responsiveness of the animals to an ovulatory dose of hCG was drastically reduced (>80% inhibition of ovulation) in the oestrogen-deprived animals; this could be overriden by exogenous administration of oestrogen. In conclusion, although blocking oestrogen synthesis in the PMSG-primed rat does not seem to alter the functional properties of the isolated granulosa cells in vitro there appears to be an effect on the number of follicles which complete maturation and are able to ovulate in vivo.

Journal of Endocrinology (1994) 142, 563–570

Administration of human chorionic gonadotrophin (HCG) or ovine LH to immature rats primed with pregnant mare serum gonadotrophin (PMSG) stimulated the rate of synthesis of polyadenylic acid (poly A)-rich RNA in the ovaries. The rate of total RNA synthesis was not affected significantly by hormone treatment, whereas protein synthesis was enhanced. The increase in the rate of synthesis of poly(A)-rich RNA in the ovaries could be inferred as induction of messenger RNA synthesis after the hormone treatment. The poly(A)-rich nature of the isolated RNA was established by oligo(dT)–cellulose chromatography, binding to Millipore filter disks and hydridization with [³H]polyuridylic acid. The level of cyclic AMP in the ovaries of such rats was also raised after administration of LH, the increase coincided with the increase in the rate of synthesis of poly(A)-rich RNA. The implications of these results are discussed in the light of the biochemical basis of luteinization and the action of LH.