The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy.

Hereditary inclusion body myopathy (HIBM; OMIM 600737) is a unique group of neuromuscular disorders characterized by adult onset, slowly progressive distal and proximal weakness and a typical muscle pathology including rimmed vacuoles and filamentous inclusions. The autosomal recessive form described in Jews of Persian descent is the HIBM prototype. This myopathy affects mainly leg muscles, but with an unusual distribution that spares the quadriceps. This particular pattern of weakness distribution, termed quadriceps-sparing myopathy (QSM), was later found in Jews originating from other Middle Eastern countries as well as in non-Jews. We previously localized the gene causing HIBM in Middle Eastern Jews on chromosome 9p12-13 (ref. 5) within a genomic interval of about 700 kb (ref. 6). Haplotype analysis around the HIBM gene region of 104 affected people from 47 Middle Eastern families indicates one unique ancestral founder chromosome in this community. By contrast, single non-Jewish families from India, Georgia (USA) and the Bahamas, with QSM and linkage to the same 9p12-13 region, show three distinct haplotypes. After excluding other potential candidate genes, we eventually identified mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene in the HIBM families: all patients from Middle Eastern descent shared a single homozygous missense mutation, whereas distinct compound heterozygotes were identified in affected individuals of families of other ethnic origins. Our findings indicate that GNE is the gene responsible for recessive HIBM.

Fecal testosterone is elevated in high ranking female ibexes (Capra nubiana) and associated with increased aggression and a preponderance of male offspring.

We monitored fecal testosterone and progesterone levels in 26 adult ibexes (17 males, 9 females) in a captive herd Nubian ibex held on 250 ha tract to test if testosterone is associated with dominance. The ibexes were observed over a 20-month period, and including two mating seasons, during which time we collected fecal samples twice during early gestation and postpartum intervals and once during lactation and pre-rut season intervals. The social hierarchy was linear with age in adult males and nearly linear in adult females. High ranking males were in solitude, but females were aggregated with the kids in the presence of a dominant female. The testosterone concentration in the males in the pre-rut (211+/-12 ng/g; N=13; dominant male 296 ng/g) was significant higher than other seasons (P<0.05). High testosterone in dominant male at pre-rut was associated with a decrease in confrontations. The individuals with the highest average testosterone concentrations were the dominant male and female (166+/-82 ng/g; 130+/-32 ng/g, N=6, respectively). In females, testosterone was highest in during the post-partum interval and was associated with an increase in aggression. The three highest fertile ranking females had higher testosterone (119+/-14 ng/g vs. 92+/-18 ng/g, P<0.05) than the four subordinate females. The sex ratio of the offspring was 8M/3F for the three older females and 5M/7F for the younger females. In early gestation period, females with only male fetuses had higher testosterone than other gravidities (119+/-14 ng/g, N=6 vs. 91+/-18 ng/g, N=7, P<0.01). Progesterone was significantly higher in the eight multiparous pregnancies than in those with the five singletons (210+/-19 ng/g vs. 186+/-12 ng/g, P<0.02). We conclude that high testosterone in females is associated with an increase in aggressive confrontations in early- and mid-lactation. In contrast, increased testosterone during pre-rut in males is associated with fewer confrontations. In addition, the data support the hypothesis that higher ranking, older dimorphic female ungulates have higher testosterone concentrations and have more male births than subordinate females.

Thoracic outlet syndrome: a multidisciplinary problem with a perspective for microsurgical management without rib resection.

BACKGROUND: Thoracic outlet syndrome (TOS) refers to a group of complex symptoms in the upper extremity caused by compression of the brachial plexus, subclavian artery and vein. Different surgical approaches were described for the management of TOS. There is, however, no "gold standard" procedure for this complicated and multidisciplinary problem. OBJECTIVES: This study evaluated the effectiveness of a microsurgical neurovascular decompression in the treatment of TOS. METHODS: 11 patients suffering from TOS (for 1.3 to 15 years after the beginning of the symptoms) were selected for a treatment of the complex symptoms of pain (diffuse or irradiated to the arm and hand), aching or paresthesia in the neck, shoulder, anterior chest, upper extremity and hand. Four of the 11 patients were suffering from signs of vascular compression. Eight patients showed slow progressive neurological deterioration (distribution of the ulnar nerve) with partial muscle atrophy. Patients underwent a microsurgical treatment using a supraclavicular approach followed by brachial plexus neurolysis, scalenectomy and release of the subclavian artery and vein without rib resection. Postoperative results were classified, using Am. J. Surg. (176: 215-218, 1998) scale (4), as good, fair and poor. RESULTS: Surgical results were studied, with a follow-up of 24 to 48 months. Prior to surgery, all patients had partial or severe limitation in physical activities. Post-operative follow-up showed that 9 (82%) of the 11 patients returned to normal everyday physical activities with a complete or significant relief of the symptoms (good results). In 2 patients (18%) the pain decreased and the use of medication was reduced (fair results). Eight of the 11 patients returned to full or partial employment. There were no cases of poor results in the study. CONCLUSION: Microsurgical neurovascular decompression of TOS without a removal of the cervical or first rib using a supraclavicular approach is an effective treatment method for a relief or complete release from symptoms and allows most patients to return to an active normal life.

Modulation of bovine placental prostaglandin synthesis by an endogenous inhibitor.

An endogenous, heat-labile, inhibitor of prostanoid synthesis in maternal caruncle tissue of bovine placentomes was studied. Inhibitory activity was present in caruncle extracts from days 120-250 of gestation, but was not detectable at term (260-280 days). The disappearance of inhibitory activity coincided with an increase in the secretion of prostanoids by dispersed caruncle cells in culture. Coculture of caruncle cells from placentomes of 120-day gestation with fetal cotyledon cells resulted in suppression of prostanoid synthesis by the cotyledon cells. However, this inhibition was not observed in cocultures of dispersed caruncle cells and fetal cotyledon cells from term placentomes. Our findings indicate that an endogenous inhibitor modulates bovine placental prostaglandin synthesis. A decline in the level of this inhibitor at term may be one factor triggering increased prostanoid synthesis required for parturition.

The interaction of cultured thyroid cells of early bovine embryos with thyrotropin and thyroglobulin.

We have prepared primary thyroid cell cultures of early bovine embryos from the first trimester of pregnancy in order to study the ontogeny of their interaction with TSH and thyroglobulin (Tg). The ability of these cells to synthesize and secrete Tg, as well as the trophic effect of TSH on the organization of the thyroid cells, were also investigated. To determine the maturation of these functions we prepared fluorescent conjugates of TSH, Tg, and anti-Tg antibodies, and visualized their interaction with the thyroid epithelial cells. Our study shows that the ability to bind TSH and Tg exists as early as the gestational age of 3 cm crown-rump length (CRL; 40 days) but does not develop linearly with embryonic age. Thus, there is a significant increase in the percentage of Tg-binding cells at 12 cm CRL, when colloid is first noticed in vivo, and a considerable elevation in TSH-binding cells around 15 cm CRL, when thyrotropic cells and TSH secretion from the fetal pituitary are first evident. Tg-containing cells and the ability to secrete Tg are observed at about 20 intrauterine days. The three thyroidal properties probably develop independently since only part of the Tg-containing cells bind Tg or TSH, and a significant proportion of the cells that exhibit Tg-binding do not bind TSH. The results support the notion that TSH is essential for the formation of follicle-like structures and effects the organization of thyroid cells into a functional structure in vitro from the late precolloidal stage.

The ontogeny of the thyrotropin-thyroid axis in early bovine embryos.

T4 and T3 formation and their response to TSH, cAMP, and prostaglandin E2 (PGE2) stimulation were studied by RIA in cultured bovine fetal thyroids from 130 embryos of 1.2-25.0 cm crown-rump length (CRL). T4 and T3 were found in all of the freshly isolated glands studied, and their concentrations increased significantly (P less than 0.05) during a 24-h incubation of glands from fetuses with a CRL greater than 8.0 cm. The release of T4 (nanograms per mg tissue), but not of T3, increased consistently with CRL (r = 0.64; P less than 0.05). The addition of TSH (0.5 mU) to the culture medium induced a 2- to 3-fold increase in the secretion of T4, but not of T3, by glands of fetuses with a CRL of 3.0-25.0 cm (P less than 0.05). Dibutyryl cAMP (10(-4) M) and PGE2 (10(-4) M) had comparable effects. A combination of TSH and theophylline (1 mM) or of TSH and dibutyryl cAMP significantly enhanced the T4 released by cultured tissue into the medium (P less than 0.05) over that induced by either agonist, but the combined effect was not fully additive. The total PGE2 in the tissue and medium was not changed by the addition of 0.5 mU TSH, and indomethacin had no effect on TSH-induced T4 secretion. The data show that thyroids of fetuses that have reached a CRL of 3.0 cm have the enzymatic capacity to produce both T4 and T3, and T4 is the dominant product formed. While exogenous PGE2 at high concentrations stimulates fetal T4 secretion, it does not mediate the actions of TSH and cAMP on the fetal thyroid.

Dexamethasone inhibition of prostaglandin production in human term placental cells is protein and ribonucleic acid synthesis dependent.

A key enzyme in the regulation of prostaglandin (PG) synthesis is PG synthase (PGS; cyclooxygenase), which converts arachidonic acid to PGs. Since both PGs and glucocorticoids are elevated before parturition, we studied the regulation of dexamethasone (DEX; 150 nM) on PGF2 alpha synthesis and PGS expression in human placental cells in vitro. Both first trimester and term placental cells were used. DEX reduced PGF2 alpha synthesis in human term placental cells, in contrast to first trimester cells which were unaffected by the same treatment. DEX inhibition of PGF2 alpha production by term placental cells was time and dose dependent. PGS expression was analyzed by [35S]methionine metabolic labeling and immunoprecipitation using polyclonal antibodies developed in rabbits against ram seminal vesicle PGS. DEX reduced PGS expression in term placental cells, but not in first trimester cells. In contrast to the effect of DEX on PGF2 alpha, progesterone and estradiol production by cells were unaffected at any stage of gestation examined. DEX inhibition of PGF2 alpha synthesis required de novo biosynthesis of RNA and proteins. These results suggest 1) corticosteroids play a role in the regulation of placental PG synthesis during parturition; 2) the inhibition of PG synthesis and PGS expression by glucocorticoids is RNA and protein biosynthesis dependent; and 3) induction of labor by glucocorticoids is not directly related to changes in placental progesterone or estradiol biosynthesis.

Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine uterine veins: LH induction of cyclooxygenase and augmentation of prostaglandin production in bovine uterine veins.

We have previously reported that bovine endometrium contains LH/human CG binding receptors and LH induces cyclooxygenase and prostaglandin production in the bovine endometrium. The present study investigated 1) whether bovine uterine vein and artery contain LH receptor messenger RNA (mRNA) and receptor protein and 2) whether LH can regulate the formation of vasoactive eicosanoids by the uterine vein. The uterine vein endothelium, but not the uterine artery, contained LH receptor mRNA transcript essentially identical to that found in the bovine corpus luteum. The uterine vein endothelium also contained a 95-kDa immunoreactive receptor protein that bound to rat anti-LH receptor antibody in Western blots. The LH receptor mRNA and LH receptor were maximally expressed in the uterine vein from cows in proestrus/estrus compared with cows in luteal or postovulatory phases. Incubation of endothelial minces of uterine vein with LH resulted in a 2-fold increase in cyclooxygenase concentration as determined by Western blot using an antibody to ram seminal vesicle cyclooxygenase. The increase in cyclooxygenase was maximal in cows in proestrus/estrus compared with postovulatory and luteal phase cows. Incubation of proestrous/estrous uterine vein or artery minces with LH or mellitin (a phospholipase A2 stimulator) caused increased production of eicosanoids. In the uterine vein, LH caused a significant increase in both PGF2alpha (basal 4.1 +/- 0.4 vs. 5.7 +/- 0.4 ng/100 mg x 6 h, P < 0.01; N = 9 cows) and PGE2 (basal 5.7 +/- 0.3 vs. 7.7 +/- 0.8 ng/100 mg x 6 h, P < 0.01; N = 6 cows) but had no effect on prostaglandin production by the artery. Mellitin increased PGF2alpha production by both uterine vein and artery minces but had no effect on PGE2 production in either tissue. Addition of steroids (progesterone, estradiol) or cytokines (tumor necrosis factor-alpha, IL-6) to the uterine vascular tissues had essentially no effect on prostanoid production. In summary, bovine uterine vein from proestrous/estrous cows expressed the LH receptor and its mRNA. Expression of the receptor may have physiological significance as LH induces cyclooxygenase and increases prostaglandin release in the uterine vein. The maximal stimulation of the receptor and its mRNA at proestrus/ estrus may serve to increase the amounts of prostanoids reaching the regressing corpus luteum either directly by increasing prostanoid production or indirectly by increasing the blood flow to the ovary.

Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12-p13.

Hereditary inclusion body myopathy (HIBM) is a group of neuromuscular disorders characterised by adult-onset, slowly progressive distal and proximal muscle weakness and typical muscle pathology. Previously, we have mapped the gene responsible for a recessive form of HIBM to chromosome 9p1 and narrowed the interval to one single YAC clone of 1 Mb in size. As a further step towards the identification of the HIBM gene, we have constructed a detailed physical and transcriptional map of this region. A high resolution BAC contig that includes the HIBM critical region, flanked by marker 327GT4 and D9S1859, was constructed. This contig allowed the precise localisation of 25 genes and ESTs to the proximal region of chromosome 9. The expression pattern of those mapped genes and ESTs was established by Northern blot analysis. In the process of refining the HIBM interval, 13 new polymorphic markers were identified, of which 11 are CA-repeats, and two are single nucleotide polymorphisms. Certainly, this map provides an important integration of physical and transcriptional information corresponding to chromosome 9p12-p13, which is expected to facilitate the cloning and identification not only of the HIBM gene, but also other disease genes which map to this region.

Granulosa cells as a source and target organ for tumor necrosis factor-alpha.

Tumor necrosis factor (TNF-alpha), a 17 kDa cytokine, is a product of activated macrophages which was recently shown to be produced by rat and bovine granulosa cells. In the present work, human granulosa cells derived from preovulatory follicles were used. It was demonstrated that human granulosa cells produce TNF-alpha (5-10 units/300,000 cells per 15 h). This production was increased by addition of follicle-stimulating hormone or by a combination of human chorionic gonadotrophin and CSF to the culture media. TNF was also found in bovine follicular fluid and the concentration was higher in the periovulatory than mid-cycle follicles. TNF-alpha was found to increase prostaglandin F-2 alpha production by human granulosa cells (P less than 0.001). We conclude that granulosa cells are both a source and target organ for TNF-alpha.

Inhibitory action of follicular fluid on progesterone and prostaglandin synthesis in bovine follicles.

The present studies examined the inhibitory effect of mid-cycle and preovulatory bovine follicular fluid (bFF) on the secretion of progesterone and prostaglandin F 2 alpha (PGF 2 alpha) by bovine theca and granulosa cells cultured in tissue culture Medium 199. The inhibitory effect of bFF on the secretion of PGF 2 alpha by incubated and cultured hemipituitary glands of male rats was also studied. Addition of 5, 10 or 20% charcoal-extracted, mid-cycle bFF to the culture medium caused a twofold decrease in the accumulation of both progesterone and PGF 2 alpha by the cultured granulosa cells (P less than 0.01). In contrast, when preovulatory bFF was used, there was no inhibitory effect on secretion of progesterone and PGF 2 alpha. The addition of 10% mid-cycle bFF to the culture medium caused a two- to fourfold decrease in the accumulation of both PGF 2 alpha and progesterone by the cultured theca (p less than 0.008). However, in the presence of 1 microgram LH/ml, the inhibitory effect of mid-cycle bFF on progesterone and PGF 2 alpha secretion was abolished. The secretion of PGF 2 alpha was significantly (p less than 0.03) decreased in male rat hemipituitary glands after 5 h of incubation or 18 h of culture. These findings suggest that bFF from mid-cycle follicles inhibits prostaglandin synthetase as well as luteinization. The inhibition disappeared with bFF from preovulatory follicles.

Effects of phyto-oestrogens on progesterone synthesis by isolated bovine granulosa cells.

The effects of phyto-oestrogens on progesterone synthesis by isolated bovine granulosa cells in vitro were assessed. Various concentrations (0.37-3700 nmol/1) of biochanin A, genistein or oestradiol were added to cultures of bovine granulosa cells and the consequent changes in progesterone synthesis were measured. The effects of alkali-soluble extracts of Rhodes grass (Chloris gayana) and fodder beet (Beta vulgaris, var.) were also examined. Another series of experiments tested the effects of genistein and oestradiol on the increase of progesterone synthesis brought about by the addition of LH to the culture medium. It was found that biochanin A and genistein increased progesterone synthesis by 40-50% when added in concentrations of 3.5 and 185 nmol/1 respectively. At higher concentrations of phyto-oestrogens greater than 176 nmol/1 for biochanin A and 1850 nmol/1 for genistein. Oestradiol showed similar effects: maximum stimulation of progesterone secretion occurred in the presence of 0.184 nmol/1; inhibition was found with concentrations of greater than 1.84 nmol/1. Extracts of Rhodes grass but not of fodder beet also showed biphasic effects. Both genistein and oestradiol in high concentrations (3700 and 3671 nmol/1 respectively) inhibited the increase in progesterone synthesis provoked by LH. The data indicated that phyto-oestrogens, like oestradiol, have a dose-related, biphasic effect on steroidogenesis in the isolated bovine granulosa cell system.

Expression of functional luteinising hormone receptor and its messenger ribonucleic acid in bovine cervix: luteinising hormone augmentation of intracellular cyclic AMP, phosphate inositol and cyclooxygenase.

The effect of luteinising hormone (LH) on bovine cervical tissues from three phases of the estrous cycle was studied. It was found that in the luteal phase cervix contained an LH receptor mRNA transcript and the 93 kDa LH receptor protein. Incubation of cervical minces from luteal phase with LH significantly increased (P < 0.05) the intracellular concentrations of LH receptor protein, cAMP, inositol phosphate (IP) and cyclooxygenase as well as the production of prostaglandin (PG) E2 (but not PGF2alpha). In contrast in the pre-oestrus/oestrus or post-ovulatory cervix, the signal for the transcript for LH receptor and the LH receptor protein was significantly lower than luteal cervix (P < 0.05) and the tissue did not respond to LH. We conclude that bovine cervix at luteal phase has high levels of LH receptor mRNA and receptor protein associated with the protein coupled receptor family that mediates cAMP and IP signalling responses. The receptors are coupled to PG synthase and generate cervical PGE2.

Functional importance of bovine myometrial and vascular LH receptors and cervical FSH receptors.

Bovine myometrium and cervix contain luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding sites, LH receptor (LH-R) messenger RNA (mRNA), and LH-R protein. Expression of LH-R is dependent on the stage of the cycle. LH-R expression is high during the luteal phase but weak during the follicular phase. In both myometrium and cervix, LH activates both the adenylate cyclase and phospholipase C pathways, and the effect of LH on both pathways at each stage of the cycle is correlated with the amount of LH-R present in the tissue. Because activation of cyclic AMP (cAMP) is associated with myometrial quiescence, we suggest that LH activation of uterine cAMP could serve to keep the uterus quiescent during the luteal phase. On the other hand, in the uterine vein LH-R mRNA and LH-R are maximal during preestrus/estrus as opposed to the luteal phase. In the uterine vein, LH increases the expression of cyclooxygenase and production of both prostaglandin E2 (PGE2) and PGF2 alpha. Because PGF2 alpha is the physiological luteolytic signal in the cow, we suggest that this increase in prostaglandin production by the uterine vein is part of the physiological events leading to luteolysis. In addition to uterine LH-R, the bovine cervix at preestrus/estrus has high levels of follicle-stimulating hormone receptor (FSH-R) and its corresponding mRNA. As with LH-R, activation of FSH-R by FSH is associated with activation of a G protein-coupled receptor family that mediates the cAMP and inositol phosphate signaling pathways. Activation of these signaling pathways is associated with an increase in the expression of cyclooxygenase and production of PGE2. Because expression of the FSH receptor was maximal at the time of the FSH peak in the blood, we suggest a physiological role for FSH in the cervix relaxation and opening at estrus.

Cytokine involvement in oocytes and early embryos.

OBJECTIVE: The early events of reproduction involve a carefully modulated complex system of oocyte maturation, fertilization, and proliferation. The aim of the study was to measure the presence of cytokines, namely interleukin 1 (IL-1), interleukin 6 (IL-6), colony-stimulating factor 1 (CSF-1), and tumor necrosis factor (TNF) in the conditioned medium (CM) of the oocytes, granulosa cells, cumulus cells, one to eight-cell embryos and sperm. DESIGN: The material was obtained from men and women undergoing in vitro fertilization therapy. MAIN OUTCOME MEASURES: We hypothesized that cytokines might affect embryonic growth and differentiation as they show a pleotropic effect on immune cells. RESULTS: All these cytokines are present in significant quantities in the CM and were shown to be expressed in a sequential manner; thus, some are present in the oocyte and its vestment, the corona-cumulus complex (IL-1, IL-6, and CSF-1), whereas TNF appears only at the stage of six to eight-cell embryos. Inflammatory cytokines could not be detected in sperm samples. CONCLUSIONS: It is possible that these cytokines have a role in the regulation of embryonic development, maternal immunological recognition of pregnancy, and maintenance of proper hormonal environment.

A possible linkage between gonadal hormones, serum and uterine levels of IgG of dairy cows.

We have investigated the possible linkage between serum and uterine fluid immunoglobulin G (IgG) levels and the hormonal status of the cow. In cycling cows there was a significant (P < 0.01) drop in average (of 4 consecutive days) serum IgG levels, from 36.4 +/- 6.7 mg ml-1 during the luteal phase of the estrous cycle to 28.3 +/- 5.3 mg ml-1 during and around estrus. In prepartum cows, there was a significant drop (P < 0.01) from an average of 37.6 +/- 3.7 mg ml-1 from 5 consecutive days, i.e. 11-7 before parturition, to 28.0 +/- 5.5 mg ml-1 on the day of parturition. Total IgG in the uterine fluid ranged from 30 to 115 mg in one horn and from 24 mg ml-1 to 70 mg ml-1 in the other horn during the luteal phase, but was essentially undetectable at estrus. The drop in serum and uterine IgG occurred concomitantly with the drop in peripheral serum progesterone, from 2-3 ng ml-1 at the luteal phase, and 11-7 days before calving to less than 0.5 ng ml-1 around estrus and calving. Data suggest a possible linkage between steroid hormone and IgG levels.

Regulation of steroidogenesis in the bovine placenta.

As pregnancy progresses in the cow, the secretory activity of the corpus luteum is markedly diminished. This reduced secretion is due to a decline in the number of viable luteal cells as well as reduction in the secretory activity and responsiveness of the cells to trophic agents. The principal extra-ovarian source of progesterone (P4) by mid-gestation therefore appears to be the placenta. Uniquely this P4 biosynthesis is cyclic-nucleotide independent, but the Ca+2 dependent. It therefore appears that the Ca+2 second messenger and protein kinase C systems are responsible for regulation of sterol biosynthesis in the cow placenta. Dispersed bovine caruncle cells from the first trimester of pregnancy in comparison to caruncle cells of older than 90 days of gestation produce little P4 and are refractory to agents which enhance placental steroidogenesis. In order to explain this refractoriness of the first trimester cells, we determine (1) the expression of P450scc and its mRNA and (2) the expression of adrenodoxin. It was found that P4 synthesis by bovine maternal caruncle cells was low or undetectable in the first trimester but increased more than 10-fold in the second trimester of gestation. Addition of 25-OH-cholesterol to second trimester maternal cells increased P5 production but no effect was observed in first trimester cells. Cytochrome P450scc and its mRNA and adrenodoxin content were determined using Western blot or dot-blot techniques. Both proteins and the mRNA were detected in maternal tissue of first and second trimesters of gestation. In conclusion low P4 levels synthesized by first trimester maternal cells are not due to the absence of either cytochrome P450scc or adrenodoxin protein or production of P450scc mRNA. The data suggest that the refractoriness of the maternal caruncle cells during the first trimester is the result of post-translational regulation.

LH/hCG receptors in the porcine uterus--a new evidence of their presence in the cervix.

High-affinity LH/hCG binding sites have been characterized in bovine, lepine, murine, human uteri and porcine myometrium and endometrium. In the present studies we analyzed these receptors in the porcine cervix. Radioreceptor ligand assays were performed with cell membrane preparations of the cervix which were analyzed for binding sites specificity, capacity and affinity. Corpus luteum and myometrium were used as positive control tissues. In the cervix there was little competition for receptor occupancy between hCG and porcine FSH (1.2%) or bovineTSH, porcine GH and porcine PRL (0.1%, 0.1% and < 0.001%; respectively) but porcine LH could completely inhibit the binding of [125I] hCG. There was not binding for LH/hCG in crude membrane preparations of kidney or skeletal muscle. The concentration (fmol/mg protein) of cervical LH/hCG receptor did not vary significantly during particular phases of the estrous cycle, except the early luteal phase (Days 6-7) when the level of LH receptors was very low (p < 0.05). The affinity of uterine LH/hCG binding sites in the cervix and the myometrium was not different from the affinity of LH/hCG binding sites in luteal cells. The porcine cervix as well as the myometrium contains a 75- and 48-kDa immunoreactive LH/cCG receptor proteins similar to corpus luteum. Southern blot of RT-PCR products performed to enhance the specificity and sensitivity of LH receptor transcripts determination in uterine tissues revealed that expected fragments of 740 and 470 bp were present in myometrium and corpus luteum. The cervix showed only 740 bp fragment. In situ hydridization showed the expression of mRNA for LH receptor in the epithelium of the cervix. Immunoreactive staining for LH/hCG receptors was also observed only in epithelial cells of the cervical tissue. Our studies are probably the first evidence demonstrating the specific LH/hCG binding sites in female cervix.

Production and regulation of progesterone in bovine corpus luteum and placenta in mid and late gestation: a personal review.

In late pregnancy the secretory activity of the corpus luteum of the cow is markedly diminished. This reduced secretion is due to a decline in the number of viable luteal cells as well as reduction in the secretory activity and responsiveness of the cells to trophic agents. The principal extra-ovarian source of progesterone in late gestation appears to be the placenta, especially the fetal cotyledon, which was shown to produce progesterone throughout gestation. Uniquely, this progesterone biosynthesis is cyclic-nucleotide independent, but Ca2+ dependent. It therefore appears that the Ca2(+)-second messenger and protein kinase C systems are responsible for regulation of sterol biosynthesis in the cow placenta.