The novel effect of hCG administration on luteal function maintenance during the estrous cycle/pregnancy and early embryo development in the pig.

Two independent experiments were performed on cyclic (Experiment I) and pregnant (Experiment II) gilts to examine the effect of human Chorionic Gonadotropin (hCG) administration on day 12 of the estrous cycle/pregnancy on ovarian and endometrial secretory function. Animals were divided into hCG Group (injection of 750 IU hCG) and Control Group (injection of saline). In Experiment I, the prolonged lifespan of the corpus luteum (CL), extended progesterone (P4) production (P < 0.05) and delayed luteolysis were found. In hCG Group increased ratio of PGE2:PGFM during 12 hrs period on day 15 (P < 0.05) of the estrous cycle was observed. In both experiments, higher concentrations of E2 in hCG treated gilts (P < 0.05) on days 14-15 of the estrous cycle/pregnancy were found. In Experiment II, hCG injection did not affect P4, PGE2 and PGFM concentrations in blood plasma, but reduced the number of resorbed embryos on day 30 of pregnancy. In the pregnant hCG treated gilts the immunostaining against von Willebrand Factor (vWF) demonstrated an enhanced (P < 0.05) angiogenesis in CLs and endometrium. Furthermore, the flow cytometry revealed an increased (P < 0.05) viability of cells in CLs of hCG Group. An augmented expression of Steroidogenic Acute Regulatory Protein (STAR; P < 0.05) and LH/hCG receptor mRNA (P < 0.05) in CLs of hCG Group were observed, but an elevated concentration of protein was confirmed only for STAR (P < 0.05). Our studies revealed, for the first time, that administration of hCG affects PGE2:PGFM ratio during the estrous cycle as well as the development of conceptuses through enhanced angiogenesis and decreased luteal apoptosis in early pregnant pigs.

Nongonadal LH receptors, their involvement in female reproductive function and a new applicable approach.

Luteinising hormone (LH) and human chorionic gonadotropin (hCG) share a common receptor in gonadal cells. The receptors have also been detected in several nongonadal but reproduction-associated tissues of pigs, cattle, and other species including the uterus (myometrium, endometrium), oviduct, cervix, blood vessels, mammary gland and other tissues. The main role of LH/hCG receptors in the myometrium is stimulation of growth and hyperplasia, and relaxation of uterine motility; hCG also boosts blood flow in the uterine artery. LH and hCG can increase production of prostaglandins in the endometrium, oviduct, and blood vessels. We suggest that the preovulatory surge of LH plays an important role in controlling oviductal contractions. Awareness of LH binding to many tissues of the female reproductive tract and integration with embryonic factors may lead to the elaboration of new strategies for improved reproductive efficiency in domestic species. Mammary glands also possess LH/hCG receptors through which gonadotropins can affect the metabolism of steroid hormones and could play an inhibitory role in mammary carcinogenesis and in the growth of breast tumours. A novel approach to target and ablate carcinoma cells through LH receptors is described.

Targeted destruction of normal and cancer cells through lutropin/choriogonadotropin receptors using Hecate-betaCG conjugate.

A recent approach to cancer treatment is destruction of malignant and non-malignant tumors by hormonally targeted lytic peptides. The presence of lutropin/choriogonadotropin (LH/CG) receptors has been confirmed in several cancer cells (e.g. breast, ovarian, and prostate). In a series of experiments conducted in vitro, we have used a conjugate of the 23-amino acid lytic peptide Hecate and a 15-amino acid segment of beta-chain of CG. To test the hypothesis that Hecate-betaCG selectively destroys porcine granulosa and luteal cells, and Leydig cancer cell line (BLT-1) possessing LH/CG receptors, the conjugate was added to culture media at different concentrations of 0.5 to 10 micro M. Spleen cells and late passage of granulosa cancer cell line (KK-1) not-possessing LH/CG receptors were used as controls. The toxicity of Hecate-betaCG conjugate was concentration-dependent in all cell types but different among various cells. The toxicity of the conjugate to treated cells was closely correlated with the number of LH/CG receptors per cell. At low concentration (1 micro M), Hecate-betaCG was more cytotoxic to cells bearing LH/CG receptors than to controls (p < 0.01). In contrast to cells possessing LH/CG receptors, cancer cell line KK-1 and spleen cells were sensitive only at concentration of 5 micro M (p < 0.001). We conclude that Hecate-betaCG selectively kills cells expressing LH/CG receptors; its toxicity is dependent on the number of binding sites for LH/CG.

Nongonadal LH/hCG receptors in pig: functional importance and parallels to human.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) share a common receptor in gonadal cells; however, the presence of these receptors has also been detected in several nongonadal but reproduction-associated tissues of pig, human, and other species. There are no data about the ontogeny of the human LH/hCG receptor. The expression of the porcine LH receptor gene in the uterus starts about 10 days after the appearance of this gene in gonads. LH/hCG receptors were found in uterus (myometrium, endometrium), oviduct, cervix, fetal membranes, and umbilical cord in humans and pigs. The main role of LH/hCG receptors in myometrium is stimulation of growth and hyperplasia and relaxation of uterine motility. hCG also increases blood flow in the uterine artery. LH and hCG can increase production of prostaglandins in endometrium, oviduct, and blood vessels. It is suggested that the preovulatory surge of LH plays an important role in controlling oviductal contractions. Human and pig mammary glands also possess LH/hCG receptors through which gonadotropins can affect the metabolism of steroid hormones in this tissue and may play an inhibitory role in mammary carcinogenesis and in the growth of breast tumors.

Distribution of NADPH-diaphorase and nitric oxide synthase (NOS) in different regions of porcine oviduct during the estrous cycle.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo- and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units +/- SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 +/- 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 +/- 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 +/- 2.5; ampulla 35.6 +/- 4.2; infundibulum 24.7 +/- 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.

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.