Clinical use of human chorionic gonadotropin in dairy cows: An update

Human chorionic gonadotropin (hCG) has a potent luteinizing hormone (LH)-like effect in cattle that extends the life span of the corpus luteum (CL) and increases progesterone synthesis, induces ovulation throughout the estrous cycle, promotes the formation of accessory corpora lutea when applied in the early luteal phase, and modifies follicular wave dynamics increasing the frequency of three-wave dominant follicular cycles. As hCG acts on ovarian cells independently of the pituitary gland and its effect is longer lasting than that produced by endogenous LH release, use of hCG rather than gonadotropin-releasing hormone (GnRH) could be targeted at populations of subfertile cows. This review describes the clinical use of hCG to improve the reproductive performance of dairy cows. In addition, we describe recent developments in the therapeutic use of hCG and studies addressing the benefits of including hCG in estrus and ovulation synchronization protocols. Our review ends with a critical discussion of how earlier findings related to ovarian responses to hCG treatment can be interpreted in the light of recent advances in the clinical applications of hCG.     

Immunocytochemical localization of oxytocin and neurophysin in human corpora lutea

Corpora lutea, corpora albicantia, and ovarian stroma from normal human premenopausal ovaries were examined for the presence of oxytocin and neurophysin by using highly specific antisera and peroxidase-antiperoxidase light-microscopic immunohistochemistry. Oxytocin and neurophysin immunoreactivity was found in some but not all cells of the corpora lutea obtained on days 19 to 24 of the menstrual cycle. Stromal tissue and corpora albicantia did not give a positive reaction for either of these peptides, and negative results were also obtained with corpora lutea of mid- and term-pregnancy and preovulatory follicles. Specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive oxytocin and neurophysin in corpora lutea of the human menstrual cycle directly demonstrates the presence of oxytocin- and neurophysin-positive cells within the human corpus luteum.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

Effect of prostaglandin E2 alpha on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF2 alpha on the hCG stimulated and basal progesterone production by human corpora lutea was examined in vitro. hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16-19 of a normal 28 day cycle), mid (days 20-22) and late (days 23-27) luteal phases. This stimulation was inhibited by PGF2 alpha (10 micrograms/ml) in corpora lutea of mid and late luteal phases. PGF2 alpha alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF2 alpha at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.     

Absence of specific follicle stimulating hormone receptors in porcine corpora lutea

Crude cell membrane preparations of corpora lutea from 13 pigs in different phases of the estrous cycle or pregnancy were assayed for the presence of specific follicle stimulating hormone (FSH) receptors using highly purified ovine FSH. Testicular tissue from boars and granulosa cells from porcine follicles served as positive controls. Scatchard analysis was used to determine binding affinity of FSH to target tissues as well to study human chorionic gonadotropin (hCG) bindability to corpora lutea membranes. In contrast to testicular tissue and granulosa cells, no specific FSH binding was detected in luteal tissue during the estrous cycle or pregnancy in pigs.     

Effect of hCG, cAMP and FSH on steroidogenesis by human corpora lutea in vitro

Human corpora lutea of various ages were minced and incubated in the presence of hCG (10 i.u./ml), cAMP (10 mM) or FSH (20 mu/ml) and production of progesterone and oestradiol was measured. Cyclic AMP and hCG stimulated progesterone and oestradiol production during at least the mid- and late luteal phases, but FSH stimulated only oestradiol production during the early and mid-luteal phases and had no effect on progesterone production. This demonstrates that progesterone and oestradiol synthesis by the human corpus luteum can be independently controlled.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.     

Luteinizing hormone receptors in ovaries of the cynomolgus monkey (Macaca fascicularis) during the menstrual cycle

Ovarian luteinizing hormone (LH) receptors were characterized using ovarian tissues from 17 cynomolgus monkeys at different phases of the menstrual cycle. Low binding affinity receptors for ¹²⁵I-LH were observed throughout the menstrual cycle. The binding affinity of these receptors for LH (< 12 × 10¹⁰ M^?1) was approximately the same as that of ovarian LH receptors previously reported in human and nonhuman primates. In addition, high-affinity receptors (17?85 × 10¹⁰ M^?1) were also detected at the mid-luteal phase, during which a large functional corpus luteum was present. Thus the high-affinity LH receptors appear with the formation of the corpus luteum and disappear with its regression. Almost no fluctuation of binding capacity was observed throughout the menstrual cycle (32?112 fmol/ mg of ovarian tissue). The high-affinity LH receptor was judged to be derived from the functional corpus luteum.     

Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.     

Effect of prostaglandins on the steroidogenesis in human ovarian tissues

Although prostaglandins are luteolytic in some species, in conditions they stimulate progesterone production in the corpus luteum (1). Apart from this effect prostaglandins may also stimulate other steps in the steroidogenic sequence e.g. corticosteroidogenesis in superfused rat adrenal glands (2) and aromatization of testosterone by perfused human placenta (3). With this possibility in view and also because of paucity of data on the effect of prostaglandins on steroidogenesis in human ovarian tissues we have been studying under conditions the effect of prostaglandins on progesterone formation in human corpora lutea and on the utilization of C₂₁ steroids by the luteal and follicular compartments of the ovary. These studies are still in progress. However, the data obtained so far indicates that in addition to stimulating progesterone synthesis in the corpus luteum prostaglandins may also affect other steps in steroidogenesis in human ovarian tissues. We wish to report here in brief these preliminary results.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.     

Effect of prostaglandin F2α on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF_2α on the hCG stimulated and basal progesterone production by human corpora lutea was examined . hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16–19 of a normal 28 day cycle), mid (days 20–22) and late (days 23–27) luteal phases. This stimulation was inhibited by PGF_2α (10 μg/ml) in corpora lutea of mid and late luteal phases. PGF_2α alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF_2α at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17beta and prostaglandin F in human corpus luteum.

Radioimmunoassay procedures have been adapted for the assay of progesterone, 17-hydroxyprogesterone, estradiol-17beta, and prostaglandin F in human corpus luteum. The method utilises a single homogenisation and extraction of the tissue followed by fractionation of the steroids on alumina, and separation of the prostaglandins of the F series from the E and A series on silica gel, prior to radioimmunoassay. An attempt has been made to validate the method for the progestins by comparison with results after fractionation of the progestins on Sephadex LH-20, for estradiol-17beta by comparison with values obtained with competitive protein-binding, and for prostaglandin F by comparison with values after additional purification. The results showed that peak concentrations of the three steroids in corpora lutea from women during the luteal phase of the menstrual cycle were comparable to those found in corpora lutea from women in early pregnancy. However, in six out of fourteen corpora lutea from non-pregnant women, prostaglandin F levels were higher than those found in corpora lutea from seven women in early pregnancy, i.e. 13-46 ng/g compared with 1-7 ng/g. Of the above six corpora lutea, four were on days 23-25 of the cycle, at a time when luteolysis would be commencing. The results in this paper support the conclusion that the corpus luteum is a major site of synthesis of the three steroids examined, although the site of synthesis of prostaglandin F is still equivocal.     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

First postpartum ovulations and corpora lutea in ewes which lamb in the breeding season

Two experiments involving crossbred ewes which lambed during the breeding season were performed to determine whether: (a) the interval to first postpartum ovulation could be reduced by weaning or mastectomy; (b) there are differences in luteal structure and luteinizing hormone (LH) receptor concentration between first postpartum corpora lutea induced with GnRH and normal cycling corpora lutea and (c) pretreatment of postpartum ewes with progesterone would affect luteal LH receptor concentration and luteal phase serum progesterone concentration.In experiment I, the mean interval (±SEM) to the first postpartum ovulation was 22.3 ± 1.1 days and was not significantly altered by weaning or mastectomy. More than half of the ewes had small, short-lived peaks of serum progesterone associated with short-lived corpora lutea prior to the normal luteal phase rise of serum progesterone. In experiment II, 2 h after GnRH injection on day 18 postpartum, serum LH concentrations were higher in ewes which received progesterone treatment on days 13 and 14 than in control ewes. Progesterone treatment did not affect mean corpus luteum weight (157 mg) or concentration of LH receptors (0.95 fmol/mg) in first postpartum corpora lutea, but progesterone-treated ewes had significantly higher endogenous serum progesterone concentrations on days 21–24. GnRH-induced corpora lutea from postpartum ewes were lighter in weight, paler in color, had lower LH receptor concentrations and had a more regressed histological appearance than corpora lutea of a similar age from normal, cycling ewes.     

Binding of high-density lipoproteins to luteal membranes: the role of prolactin, luteinizing hormone, and circulating lipoproteins

Ovarian and adrenal membranes from immature gonadotropin-primed rats, treated with 4-amino-pyrazolopyrimidine (4APP) to reduce endogenous lipoprotein levels, displayed higher binding of porcine high-density lipoprotein (HDL) when compared to control rats. Immature, hypophysectomized (HYPOX) rats bearing corpora lutea (CL) on Day 5 after ovulation had lower levels of serum progesterone and reduced capacity for HDL and human chorionic gonadotropin (hCG) binding to ovarian membranes when compared with intact animals. Hypophysectomy also reduced the number of HDL binding sites in adrenal membranes. Treatment of HYPOX animals with luteinizing hormone (LH) and prolactin (Prl) alone or in combination increased the HDL binding sites in the ovary relative to HYPOX-untreated rats. Neither hormone affected binding to adrenals, where only adrenocorticotropic hormone (ACTH) enhanced HDL binding. LH treatment reduced the serum progesterone levels and hCG binding to the ovaries, whereas Prl administration increased progesterone levels with no effect on hCG binding. We conclude from this study that HDL binding in the luteinized ovary is regulated by Prl and LH and circulating lipoproteins, whereas in adrenals it is regulated by ACTH and circulating levels of lipoproteins.     

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.     

Evidence for a gonadal nonsteroidal factor that specifically inhibits release of luteinizing hormone in ewes.

Bilaterally ovariectomized ewes were used to investigate the effect of systemic administration (i.v.) of charcoal-treated aqueous luteal extracts from ovine corpora lutea on plasma concentrations of pituitary gonadotrophins. Jugular blood samples were taken every 15 min at least 5 h before (control period) and 5 h after (treatment period) injection. In Expt 1, the administration of luteal extract from corpora lutea of days 70-76 of pregnancy, but not of the extract prepared from muscular tissue, resulted in a significant decrease of mean concentrations of luteinizing hormone (LH) (P < 0.02) and frequency of LH pulses (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations were not affected by injections of either extract. These findings provide the first demonstration of the presence of a nonsteroidal factor in the corpus luteum of midpregnancy that selectively suppresses the secretion of LH. In Expt 2, mean concentrations of LH and FSH and frequency of LH pulses were unaffected by injections of luteal extracts from ovine corpora lutea of days 10-12 of the oestrous cycle or day 15 of pregnancy. These data suggest that some factor(s), probably from the fetoplacental endocrine unit, is required to ensure the production of a significant quantity of the luteal LH-inhibiting factor after day 15 of pregnancy. In Expt 3, treatment of luteal extract from corpora lutea of day 70 of pregnancy with proteolytic enzymes destroyed the LH-inhibiting activity, suggesting the proteic nature of the luteal LH-inhibiting factor. In Expt 4, plasma concentrations of LH were not affected by injection of charcoal-treated extract prepared from fetal cotyledonary tissue of days 110-120 of pregnancy suggesting that the LH-inhibiting factor exclusively originates from the corpus luteum during pregnancy. These experiments provide the first direct evidence for the existence of a potent nonsteroidal factor of luteal origin that specifically inhibits pulsatile secretion of LH, without influencing FSH release in female animals. We propose the term LH-release-inhibiting factor (LH-RIF) to describe this activity.