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Vitellogenin detection and chick pathology are useful endpoints to evaluate endocrine-disrupting effects in avian one-generation reproduction study.

To investigate additional endpoints for screening of endocrine disruptors in birds, effects of 17beta-estradiol (E2) on one-generation reproduction in the Japanese quail (Coturnix japonica) were assessed. Pairs of the 10-week-old Japanese quail were fed a low-phytoestrogen diet containing E2 at 0 (control), 10, 100, and 1,000 ppm for six weeks. In the E2 100- and 1,000-ppm groups, the parental quail represented marked toxic changes including high mortality, decreased food consumption, decreased gonad weights, gross and histologic toxic changes in the reproductive and other organs, and inhibition of the reproduction. However, no adverse effects were observed in the parental quail from the E2 10-ppm group. In the parental males, serum vitellogenin (VTG) concentrations were increased significantly in the E2 10-ppm group, disclosing that serum VTG concentration is one of the highly sensitive endpoints for evaluating estrogenic endocrine activities. In the E2 10-ppm group, number of eggs laid, number of eggs with abnormalities, eggshell strength and thickness, fertility, early and late viabilities of embryos, normal hatchling rate, and clinical signs, mortality, viability, and body weight of chicks at 14 d of age were not affected. However, histopathology of the chicks in the E2 10-ppm group revealed meaningful morphological changes in the reproductive organs, such as cystic dilatation of seminiferous tubules, increased interstitial cells in the testis, and decreased theca cells in the ovary. The present study suggests that serum VTG concentration in the parental quail and histopathology of reproductive organs in the offspring are sensitive endpoints and are useful as additional endpoints in the avian one-generation reproduction test using the Japanese quail for evaluating estrogenic endocrine-disrupting effects.

Animals↗

Estrogen receptor alpha and beta expression in the porcine ovary.

In order to investigate the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the porcine ovary, in situ hybridization was applied. Specific ovine ERalpha and bovine ERbeta cDNA probes were labeled with [-32S]dCTP. In the porcine ovary, positive signals for ERbeta were found in both granulosa and theca cells of all types of antral follicles as well as in the corpora lutea at all stages of regression. ERalpha mRNA was limited exclusively to the granulosa cells of preovulatory follicles and was present in a few cells of early corpora lutea. Thus, we showed differential expression of ERalpha and ERbeta at the mRNA level. Large antral follicles and early corpora lutea are the site, where both forms of estrogen receptor are expressed.

Animals↗

Changes of messenger RNA expression of angiogenic factors and related receptors during follicular development in gilts.

The interaction between angiogenic factors and related receptors is closely associated with follicular angiogenesis. The present study was performed to determine the relationships between the capillary network and mRNA expression of several angiogenic factors and related receptors during porcine follicular development. Ovaries in gilts were collected 72 h after eCG (1250 IU) treatment for histological observation. Granulosa cells and thecal tissues in small (diameter, <4 mm), medium (diameter, 4-5 mm), or large (diameter, >5 mm) individual follicles were collected for detection of mRNA expression of vascular endothelial growth factor (VEGF) 120, VEGF 164, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in granulosa cells and fms-like tyrosine kinase (Flt-1), fetal liver kinase (Flk-1) or the murine homologue of kinase domain region (KDR), bFGF receptor (bFGF-R), and EGF receptor (EGF-R) in thecal tissue by semiquantitative reverse transcription-polymerase chain reaction. The eCG treatment resulted in the emergence of healthy preovulatory follicles (diameter, >6.0 mm) that possessed more capillaries in the thecal cell layer and a significant increase in the percentage of atretic follicles of 1.0-2.9 mm in diameter. The number of capillaries in the thecal cell layer increased significantly in healthy follicles larger than 3 mm in diameter in the eCG group compared with those in controls. The expression of VEGF 120, VEGF 164, and bFGF mRNAs increased in granulosa cells of medium and large follicles from ovaries of prepubertal gilts after eCG treatment. The Flt-1, Flk-1/KDR, and bFGF-R mRNA expression increased in theca cells of medium and large follicles after eCG treatment. The expression of EGF mRNA increased in granulosa cells of small, medium, and large follicles from ovaries after eCG treatment, but the mRNA expression of EGF-R in thecal tissue did not change. These data indicate that preovulatory follicles possessed a larger capillary network and expressed more mRNAs of angiogenic factors in granulosa cells and related receptors in thecal tissue. We concluded that VEGF 120, VEGF 164, bFGF, and EGF may be greatly involved in the angiogenic process of follicular development in prepubertal gilts with eCG treatment.

Animals↗

Alpha 6 subunit of integrins in the development and sex differentiation of the mouse ovary.

The localization of the alpha 6 subunit of integrins in the ovary was studied by conventional and immunolabeling light and electron microscopy starting from the pregonadal embryonic phase until adulthood. The formation of gonadal blastema cells included an initial expression of the alpha 6 subunit on the plasma membranes of all blastema cells. Subsequently the reaction for the alpha 6 subunit became restricted in groups of these cells, which differentiated into gonadal cord cells, the precursors of follicular cells. The alpha 6 subunit was also found in the cells of the mesonephric duct, mesonephric tubules, and the ovarian rete. Reorganization of the gonadal cords into follicles at birth was accompanied with strong and uniform re-expression of the alpha 6 subunit on the surface of the cord cells. Vascular endothelial cells and the cells of the postnatal surface epithelium remained positive for the alpha 6 integrin subunit. In larger follicles, the intensity of the reaction for the integrin subunit varied. The theca cells of growing follicles contained the alpha 6 subunit. The results show that this subunit of integrins is present in phases of increased adhesion and aggregation, and that its expression probably is involved in the regulation of ovarian epithelial differentiation. The distribution of alpha 6 integrin in ovarian cells shows potentially important sex-specific and developmental differences in epithelial organization when compared with respective changes found earlier by us in the male gonad.

Animals↗

Lectin staining studies on follicular atresia in house rat (Rattus rattus).

Topographic distribution of terminal and intercalary carbohydrate moieties were studied using horseradish peroxidase conjugated Glycine max (GMA), Arachis hypogea (PNA), Lotus tetragonolobus (LTA), Bandirarea simplicifolia (BSA), and Dolichos biflorus (DBA) agglutinins. N-Gal NAc, alpha-D-Gal, alpha-L-Fuc, D-Gal (beta-1-3)-D-Gal NAc, and alpha-beta-D Gal NAc linked glyco-conjugates were exposed during follicular atresia in oocyte, zona pellucida and granulosa cells. Theca interna and interstitial gland tissue revealed homology in lectin binding.

Agglutination↗

Immunoreactive corticotropin-releasing hormone and its binding sites in the rat ovary.

Corticotropin-releasing hormone (CRH), the principal neuropeptide regulator of pituitary ACTH secretion, is also produced at peripheral inflammatory sites, where it acts as a proinflammatory cytokine, and by the Leydig cell of the testis, where it exerts autocrine inhibition of testosterone biosynthesis. Because key ovarian functions, such as ovulation and luteolysis, represent aseptic inflammatory responses, and because the theca cell is the functional equivalent of the Leydig cell, we explored the CRH presence in the ovary, first, by specific CRH immunohistochemistry of adult cycling female Sprague-Dawley rat ovaries. We detected cytoplasmic immunoreactive CRH (IrCRH) in theca and stromal cells and in cells within the corpora lutea, at all phases of the estrous cycle. Using a specific radioimmunoassay, we measured IrCRH in extracts of rat ovaries (0.042-0.126 pmol/g wet tissue). The mobility of the ovarian IrCRH molecule was similar to that of rat/human CRH by reverse phase HPLC. To investigate the CRH action in the ovary, we identified, characterized, and localized CRH receptors in the rat ovary. Binding was linear with increasing tissue concentration, saturable, and of high affinity. Scatchard analysis of 125I-Tyr-ovine CRH competitive displacement curves indicated a high affinity binding site with a Kd of approximately 6 nM and a Bmax value of approximately 61 fM/mg protein. Autoradiographic studies revealed CRH receptors primarily in ovarian theca and stroma. We conclude that IrCRH and CRH receptors are present in rat ovaries, suggesting that this neuropeptide may play a regulatory role in this gonad, perhaps through its proinflammatory properties and/or by participating in the auto/paracrine regulation of steroid biosynthesis. Functional studies are necessary to define the role(s) of CRH in the ovary.

Animals↗

Paracrine regulation of ovarian granulosa cell differentiation by stanniocalcin (STC) 1: mediation through specific STC1 receptors.

Stanniocalcin (STC) in fish maintains calcium and phosphate homeostasis, whereas mammalian STC1 shows a diverse tissue expression pattern with ovary exhibiting the highest level. Based on the known expression of STC1 in theca/interstitial cells of the ovary, we generated recombinant N-glycosylated STC1 protein and tested its ability to modulate granulosa cell differentiation. In cultured rat granulosa cells obtained from early antral follicles, treatment with STC1 suppressed FSH-stimulated progesterone biosynthesis with minimal effects on estradiol and cAMP production. In mature granulosa cells, treatment with STC1 also suppressed human chorionic gonadotropin-induced progesterone production. The inhibitory effect of STC1 was accompanied by a pronounced suppression of the CYP11A transcripts and the FSH induction of functional LH receptors. In addition, STC1 was found to act downstream of adenyl cyclases in suppressing progesterone biosynthesis. We also tested the regulation of STC1 gene expression by gonadotropins. Treatment with pregnant mare serum gonadotropin decreased STC1 transcript levels in theca cells of maturing follicles, whereas subsequent treatment with human chorionic gonadotropin led to sustained suppression in the corpora lutea. Using radiolabeled recombinant STC1, receptor assays showed specific STC1 binding with a high affinity to granulosa cells. Because STC1 is expressed in ovarian theca/interstitial cells, the present demonstration of receptor binding and the specific actions of STC1 in granulosa cells suggest the existence of a follicular paracrine system in which theca cell-derived STC1 dampens the gonadotropin stimulation of granulosa cell differentiation. The observed STC1 suppression of progesterone, but not estradiol, production further suggests the potential role of this paracrine hormone as a luteinization inhibitor.

Animals↗

Inositol phosphate stimulation by LH requires the entire alpha Asn56 oligosaccharide.

Lentil lectin-bound, fucose-enriched hTSH was reported to stimulate both cAMP and inositol phosphate (IP) intracellular signalling pathways, whereas fucose-depleted hTSH stimulated only the cAMP pathway. Gonadotropins activate the cAMP pathway and in several studies higher concentrations activate the IP pathway. Since only the 10% of alpha subunit Asn(56) oligosaccharides (Asn(52) in humans) are fucosylated, the higher glycoprotein hormone concentrations required for IP pathway activation might be related to the abundance of competent hormone isoforms. Lentil lectin-fractionated equine (e)LHalpha and eFSHalpha preparations were combined with a truncated, des(121-149)eLHbeta preparation. All four hybrid hormone preparations induced IP accumulation in porcine theca cells, suggesting that activation of the IP pathway was not dependent on fucosylation at alpha subunit Asn(56). However, the presence of Asn(56) carbohydrate was necessary for increased IP accumulation. Intact, rather than Asn(56)-deglycosylated eLH preparations provoked a biphasic steroidogenic response by rat testis Leydig cells, suggesting that Galpha(i) stimulation was also sensitive to loss of Asn(56) carbohydrate. While rat granulosa cells responded to human FSH preparations in a biphasic manner, a classical sigmoidal response was obtained to eFSH and Asn(56)-deglycosylated eFSH, suggesting that the equine preparations did not activate Galpha(i). Purified oLHalpha Asn(56) oligosaccharides inhibited FSH-stimulated steroidogenesis in rat granulosa cell cultures indicating a direct role for carbohydrate in FSH action. The same carbohydrate preparation inhibited hCG-stimulated fluorescence energy transfer suggesting oligosaccharide involvement in activated LH receptor self-association.

Animals↗

Testosterone synthesis by chicken follicular cells.

The roles of theca and of granulosa cells in the synthesis of testosterone (T) in ovarian follicle of hens have been studied. Theca cells alone synthesized no progesterone (P), and no or only negligible amounts of T. Granulosa cells, in contrast, without stimulation produced large amounts of P and small amounts of T. When incubated with ovine gonadotrophins, theca cells showed greater response than granulosa cells in synthesizing more T (2- to 2.5-fold increase). Combination of the two follicular cell types decreased synthesis of P, but increased that of T (9- to 17-fold). When 0.1 microgram of ovine luteinizing hormone (oLH) was added to the combined cells, T concentrations increased most dramatically, a 83-fold increase was seen after 12 h of incubation. Theca cells incubated with exogenous P also produced large amounts of T. These results indicated that in hens, theca cells may be the major site of follicular T synthesis, however, theca cells alone cannot synthesize T unless granulosa cells or P were added. It is suggested that granulosa cells may use P as a precursor, and that P is converted to T in the theca cells.

Animals↗

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization.

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.

Animals↗

An immunohistochemical label to facilitate counting of ovarian follicles.

U.S. and internationally harmonized Health Effects Test Guidelines for Reproduction and Fertility Effects include enumeration of primordial and developing ovarian follicles as endpoints of safety tests, and the number of these structures is also of interest for other aspects of reproductive biology. Performing the counts microscopically on representative hematoxylin and eosin (H&E)-stained sections of ovary is tedious and error-prone. The ability to mark oocyte nuclei distinctly with an antibody significantly increases speed and accuracy of counting. We have identified a rabbit polyclonal antibody directed against a synthetic 14-amino acid sequence from human cytochrome P-450 1B1 (CYP1B1) that unequivocally marks rodent oocyte nuclei, in addition to nuclei of some ovarian granulosa and theca cells. Follicles of all degrees of maturity are easily distinguished from ovarian background; ability to detect and identify primordial follicles is particularly enhanced. High-contrast and high-resolution labeling was achieved with routine immunohistochemical procedures using an avidin-biotin-peroxidase method on rat and mouse tissues fixed in 10% neutral buffered formalin.

Animals↗

[Role of local peptide regulators in the maturation of ovarian follicles].

Follicular maturation and ovulation are triggered by pituitary hormones, specifically, follicle stimulating hormone (FSH) and luteinizing hormone (LH). Nevertheless ovarian response to gonadotropins is systematic and not targeted. Adequate physiological response, i.e. maturation of a single dominant follicle and a single ovulation, is dependent on local modulation of the response by local paracrine and autocrine regulators. Presently, we may conclude TGF alpha and IGF-I and II are implied in growth of cellular components of the follicles: granulosa and theca cells. TGF beta and activins stimulate cellular differentiation with production of estrogens and inhibins. TGF alpha is the single local factor which accelerates the meiotic competence acquisition. Finally, follistatin seems to be a powerful atretic factor for cohort follicles and luteinizing agent for dominant follicle.

Activins↗

The expression of cyclin-dependent kinase inhibitors p15, p16, p21, and p27 during ovarian follicle growth initiation in the mouse.

BACKGROUND: Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs). CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs). METHOD: In this study we tested the expression of CDKIs p15, p16, p21 and p27 by immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16). RESULTS: Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle stage. However, p16 staining was stronger (++) in the oocytes of all primordial, and 57.4 +/- 3.1% of primary follicles compared to the remaining primary and more advanced follicles (+). Interestingly, primary follicles with weaker (+) oocyte staining for p16 had significantly larger mean follicle diameter compared to the primary and primordial follicles with stronger (++) oocyte staining (55.6 +/- 2.1 vs. 32.0 +/- 1.0 and 26.5 +/- 0.7 microm, respectively, p < 0.0001). This difference in follicle diameter was mainly due to a larger mean oocyte diameter (primary follicles, stronger vs. weaker, 19.6 +/- 0.6 vs. 31.5 +/- 1.4 microm, p < 0.0001). Oocytes of atretic follicles showed stronger staining with all four CDKIs. CONCLUSIONS: These preliminary findings suggest that the initiation of oocyte growth, which seems to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further studies are needed to investigate the factors that regulate the expression of p16 in the oocyte, which might also govern the initiation of primordial follicle growth.

Animals↗

Is irregular regression of corpora lutea in climacteric women caused by age-induced alterations in the "tissue control system"?

PROBLEM: We have recently observed that the regression of corpora lutea (CL) in women during the reproductive period of life is accompanied by a diminution of Thy-1 differentiation protein release from vascular pericytes and an accumulation of T lymphocytes and activated macrophages among both degenerating granulosa lutein cells (GLC) and theca lutein cells. These data suggest that the immune system and other stromal factors, representing components of the "tissue control system," may play a role in regression of the CL. We investigated degenerating CL from climacteric women to address the possibility that the decline of immune functions with advancing age may result in incomplete regression of luteal tissue. This could contribute to the altered hormonal profiles and abnormal uterine bleeding that frequently occur during the climacteric. METHOD: Immunoperoxidase staining and image analysis were used to localize Thy-1 differentiation protein of vascular pericytes, cytokeratin staining of GLC, neural cell adhesion molecule expression by theca lutein cells, CD15 of neutrophils, CD4, CD14, CD68, and leukocyte common antigens of macrophages, and CD3 and CD8 determinants of T lymphocytes. We also investigated the expression of luteinizing hormone receptor (LH receptor) and mitogen activated protein kinases (MAP kinases) in luteal cells. Samples of regressing luteal tissue were obtained during the follicular phase from perimenopausal women (age 45-50) who exhibited prolonged or irregular cycles. For comparison, luteal tissues from women with regular cycles (age 29-45) and CL of pregnancy were also investigated. RESULTS: Corpora lutea of the climacteric women exhibited irregular regression of luteal tissue characterized by a lack of cytoplasmic vacuolization and nuclear pyknosis in GLC, and by a persistence of theca lutein cells exhibiting hyperplasia and adjacent theca externa layers. This was accompanied by a continuing release of Thy-1 differentiation protein from vascular pericytes. Persisting GLC lacked surface expression of macrophage markers (CD4, CD14, CD68 and leukocyte common antigen) as well as nuclear granules exhibiting CD15 of neutrophils, detected in regularly regressing GLC. In addition, such persisting GLC showed weak or no LH receptor expression, and retained the expression of cytokeratin. They also exhibited enhanced staining for MAP kinases. Strong cytoplasmic MAP kinase expression with occasional nuclear translocation was also detected in persisting theca lutein cells, indicating high metabolic activity of these cells. T lymphocytes, although occasionally present in luteal stroma within luteal convolutions, did not invade among persisting GLC and were virtually absent from layers of theca externa and theca lutein cells. CONCLUSIONS: These data indicate that the regressing CL in climacteric women may exhibit persistence of luteal cells, perhaps because of age-induced alterations of the immune system and other local stromal homeostatic mechanisms involved in the elimination of luteal cells. Persisting GLC and/or theca lutein cells may exhibit abnormal hormonal secretion that contributes to the alteration of target tissues, such as the endometrium, resulting in abnormal uterine bleeding, hyperplasia, and neoplasia.

Adult↗

Reproductive biology of the relaxin-like factor (RLF/INSL3).

The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.

Amino Acid Sequence↗

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine theca and granulosa cells.

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors - proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) - are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15-16 of the estrous cycle) and large (days 19-20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO(2), in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (beta-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.

Animals↗

Luteal phase defect: a review of pathophysiology.

Corpus luteum function depends on normal granulosa and theca cell components, which in turn are stimulated by an adequate luteinizing hormone (LH) surge in both duration and amplitude of pulses with LH residual pulses of adequate amplitude during the 14-day luteal span. The granulosa component must be competent to 1) synergize with thecal androgen production for increased estrogen production, 2) develop LH receptors for progesterone production, and 3) mature the egg cytoplasmically. The theca component must be capable of inducing angiogenesis factor to increase follicular blood supply and must be responsive to an LH pulse and human chorionic gonadotropin for corpus luteum rescue. The corpus luteum defect with a normal 14-day span is related either to an inadequate granulosa cell or to an inadequate LH surge, but a fairly normal LH pulse and theca cell response. The short luteal phase defect is related to a poor LH surge and an absent or extremely poor LH pulse. Diagnostic studies have indicated that the endometrial biopsy is the most efficient diagnostic method. Severe luteal defects can be diagnosed by a progesterone assay if the entire cycle is assayed, but single or even multiple progesterone assays are unreliable. The etiology is multifactorial and usually is related to the hypothalamic-pituitary factors influencing the LH surge, rather than to ovarian factors. Factors may vary from cycle to cycle, making it important to determine that the defect is repetitive. The specific etiology is often difficult to determine; substitution progesterone therapy is the most satisfactory treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise↗