Late Follicular Stage Research Articles

Insulin-like growth factors and their binding proteins in the ovine oviduct during the oestrous cycle.

The oviduct is the site of fertilization, and factors present in the oviductal fluid appear to be crucial to the future success of conceptus development. The spatial and temporal localization of mRNA encoding components of the insulin-like growth factor (IGF) system (IGF-I, IGF-II, the type 1 IGF receptor, and IGF-binding proteins -2, -3 and -4) in the ovine oviduct were examined in tissue samples taken during the early and late stages of follicular development, and the early, mid-, and late luteal phases using in situ hybridization. Expression of mRNA encoding IGF-I showed a cyclical pattern, increasing sharply in the mucosa and muscularis during the late follicular phase (P < or = 0.001), then declining. In the muscularis, mRNA encoding IGF-II exhibited no temporal changes, but concentrations in the mucosa increased from the late follicular stage to the early luteal phase. mRNA encoding the type 1 IGF receptor was present throughout the oviduct. Concentrations increased during the follicular phase to peak in the early luteal phase in both the mucosa and muscularis (P < 0.001). IGFBP-2 gene transcripts were undetectable at all time points examined. mRNAs encoding IGFBP-3 and IGFBP-4 were localized primarily in the stromal region. IGFBP-3 expression peaked in the late follicular stage of the cycle P < or = 0.001). The concentration of mRNA encoding IGFBP-4 increased in the follicular phase and was maintained at a significantly higher concentration during the early and mid-luteal stages (P < 0.001). The co-ordinate maximum expression of mRNA for both IGF-I and IGF-II, the type 1 IGF receptor and IGFBP-3 during the period when the gametes and embryo are in transit suggests a role for IGF-I and possibly IGF-II peptides in providing an oviductal environment propitious to conception and early embryonic growth and metabolism.

Regional distribution and hormonal control of estrogen-dependent oviduct-specific glycoprotein messenger ribonucleic acid in the baboon (Papio anubis).

Our objective in this study was to complete the sequence of the baboon oviductal glycoprotein, examine the hormonal regulation of the oviductal glycoprotein mRNA, and determine whether there was a regional variation within the oviduct in the level of oviductal glycoprotein mRNA expression. Finally, because of the structural similarity of the amino terminal end of the oviductal glycoprotein to chitinases, we sought to determine whether the oviductal glycoprotein functions as a glycosyl hydrolase. The total transcript length of the baboon oviductal glycoprotein was determined to be 2228 nucleotides in length plus a poly(A) tail. The largest open reading frame was 623 amino acids, which would produce a protein of 69.3 kDa. The first 420 amino acids were highly homologous to the amino acid sequence of other oviductal glycoproteins, but the remainder of the sequence differed considerably from that of all other species except the human. Although the N-terminal region exhibited sequence similarity to chitinases, the oviductal glycoprotein did not exhibit any activity towards typical chitinase substrates. The oviductal glycoprotein mRNA levels were elevated to approximately the same extent in the fimbria, ampulla, and isthmus of the oviduct after estradiol treatment and in the late follicular stage of the menstrual cycle. The oviductal glycoprotein mRNA levels were lower in the early follicular stage and early luteal stage and were not detectable in the late luteal stage or in progesterone-treated baboons. These results indicate that the oviductal glycoprotein mRNA is induced by estradiol and is present at the highest levels at the time of fertilization. Although there is structural homology with chitinases, no such glycosyl hydrolase activity could be detected. However, the common structure of the N-terminal region of the oviductal glycoproteins implies that it has the same, as yet unknown, function in all species.

Menstrual cycle specific expression of epidermal growth factor receptors in human fallopian tube epithelium

We studied the expression of epidermal growth factor (EGF) receptor protein and messenger RNA (mRNA) in human fallopian tubes at three stages of the menstrual cycle: early follicular (n = 3), late follicular (n = 3) and luteal (n = 3). Immunohistochemical studies in the ampullary portion of the tubes showed that specific staining was localized to the epithelium and the vascular endothelium. Staining of the epithelium was intense at the late follicular and luteal stages, while it was weak at the early follicular stage. 125I-EGF binding study in the tubal plasma membranes revealed a class of high-affinity EGF receptors. Although dissociation constants were similar between the stages, numbers of binding sites at the late follicular and luteal stages were significantly (P < 0.01) greater than those at the early follicular stage. Western blotting showed that tubal plasma membranes contain M(r) 170,000 EGF receptor protein. The amounts were significantly (P < 0.01, n = 3) greater at the late follicular and luteal stages than those at the early follicular stage. Reverse transcription and polymerase chain reaction (RT-PCR) revealed that EGF receptor mRNA was expressed in all the 9 RNA samples (n = 3 for each stage) from the tubal ampullary portion. The amounts were significantly (P < 0.01, n = 3) greater at the late follicular and luteal stages than those at the early follicular stage (by a competitive PCR). Increase in the amounts of EGF receptor protein and mRNA occurred in association with an increase in serum oestradiol but not progesterone levels. Next we examined whether EGF receptor and its ligands (EGF and transforming growth factor alpha) are directly induced by oestrogen. We found that specific staining for EGF receptor and its ligands in the tubal epithelium was detected (by immunohistochemistry) in postmenopausal women with oestrogen replacement (n = 3), but not in subjects without oestrogen replacement (n = 3). These results suggested that EGF receptors in the human tubal epithelium are expressed in relation to specific stages of the menstrual cycle and that the expression may be induced by oestrogen.

Changes in Epidermal Growth Factor Receptor and the Levels of its Ligands during Menstrual Cycle in Human Endometrium

We examined menstrual cycle-dependent changes in the expression of human endometrial epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and epidermal growth factor receptor (EGFR) and their mRNA using immunoblot analysis, 125I-EGF binding, and competitive reverse transcription and polymerase chain reaction (RT-PCR). We also studied their localization in the endometrial tissue by immunohistochemistry. Endometrial samples were obtained at three stages of menstruation: the early follicular stage, which exhibits low serum estradiol (E2) and progesterone (P) levels; the late follicular stage, which exhibits high E2 and low P levels; and the luteal stage, which exhibits high E2 and P levels. Immunohistochemical examination showed that EGF, TGF alpha, and EGFR were localized to the endometrial epithelium. Immunoblot analysis revealed that endometrial EGF, TGF alpha, and EGFR levels were significantly (p < 0.01) increased at the late follicular and luteal stages compared to the early follicular stage. 125I-EGF-specific binding levels at the late follicular and luteal stages were significantly (p < 0.01) higher than at the early follicular stage, consistent with the results of immunoblot analysis. Competitive RT-PCR revealed that EGF, TGF alpha, and EGFR mRNA levels were significantly (p < 0.01) higher at the late follicular and luteal stages than at the early follicular stage. Changes in EGF, TGF alpha, and EGFR mRNA levels were consistent with changes in protein levels. These findings suggest that synthesis and expression of human endometrial EGF, TGF alpha, and EGFR vary with the stage of the menstrual cycle and that their expression in the human endometrium is associated with the increase in the serum E2 but not with the increase in P levels.

Complementary Deoxyribonucleic Acid Cloning and Molecular Characterization of an Estrogen-Dependent Human Oviductal Glycoprotein1

A 120-kDa oviduct-specific glycoprotein is synthesized and secreted into the oviductal lumen during estrogen dominance in the human. The objective of this investigation was to clone, sequence, and characterize the cDNA to this core protein. Rapid amplification of cDNA ends was used to clone a contiguous 3' CDNA end and 5' cDNA end. The total length of the cDNA was determined to be 2.2 kb by sequence analysis and exhibited a 92% sequence identity with the comparable overlapping baboon cDNA (1.2 kb). A high degree of homology was found to the N-terminal sequence of hamster oviductin and the partial sequence of a homologous baboon and bovine oviduct glycoprotein. Northern blots revealed a single mRNA species of 2.4 kb. Using RNA from various tissues of an estrogen-treated baboon, we found that the mRNA for the oviductal glycoprotein was present only in the oviduct. Hybridization was detected to an mRNA of similar size from oviductal tissue of the baboon, hamster, and mouse and to an mRNA of slightly smaller size in the rabbit, cow, and cat but not to any mRNA species from rat oviductal RNA. Slot-blot analysis showed that the message was present in significantly greater (p < 0.05) concentrations in RNA from oviductal tissue from the late follicular stage than from the early follicular, early or late luteal, or postpartum stages. In conclusion, we have isolated the complete cDNA for a human oviductal glycoprotein. The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control. The mRNA for the oviductal glycoprotein is present only in the oviduct of an estrogen-treated baboon, and a cross-hybridizing RNA is found in oviductal RNA from various mammals.

Expression of epidermal growth factor and transforming growth factor-alpha in fallopian tube epithelium and their role in embryogenesis.

We studied the expression of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha in human fallopian tube epithelium at various menstrual stages. Immunohistochemical staining using anti-EGF and anti-TGF-alpha antibodies showed a specific staining in ampullary tube epithelium at late follicular and luteal stages but the staining was very weak at the early follicular stage. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) using beta-actin mRNA as an internal standard revealed the menstrual-stage-specific expression of EGF and TGF-alpha gene transcripts: amounts of EGF and TGF-alpha mRNA relative to those of beta-actin were significantly higher at late follicular and luteal stages than at the early follicular stage. To clarify the biological role of these growth factors, mouse 2-cell embryos were cocultured with human fallopian tube epithelial cells with or without blocking the action of these growth factors. Cocultures significantly promoted blastocyst formation, but this promotive effect of the tubal epithelial cells was completely abolished by the addition of anti-EGF and/or anti-TGF-alpha monoclonal neutralizing antibodies to the coculture system. These results demonstrated that EGF and TGF-alpha were synthesized and expressed in fallopian tube epithelium at specific menstrual stages, and may be involved in early embryonic development.

Correlation of follicular steroid hormone profiles with ovarian cyclicity in sows.

Ovaries were obtained from sows immediately after slaughter and were morphologically assigned to different stages of the ovarian cycle. Follicular fluid contained in the predominating follicles was analysed for ten steroid hormones by a multiple, simultaneous radioimmunoassay technique. The steroids measured were pregnenolone, progesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone, oestrone and oestradiol-17 beta. The concentrations of the steroids remained relatively low during the luteal stages until the mid-follicular stage when ovaries contained predominantly small to medium-sized (less than 5.0 mm in diam.) follicles. With the exception of 5 alpha-dihydrotestosterone concentrations, which remained low regardless of the size of the follicles or the stage of the cycle, the concentrations of all the steroids were significantly elevated in the transition from the mid- to late follicular stage, a period when the ovaries contained mainly large (6-10 mm diam.) follicles. Follicles at the ovulatory stage exhibited a profound decline in the concentrations of androgens and oestrogens. In contrast, the magnitude of decline in the levels of 3 progestagens, i.e. pregnenolone, progesterone and 17 alpha-hydroxyprogesterone, was much less than that for androgens and oestrogens, while the concentration of 20 alpha-dihydroprogesterone was actually elevated in the ovulatory follicles. The present results agree with those of earlier studies which measured fewer steroids in follicles obtained by repeated and sequential laparotomies of sows during spontaneous cycles. In contrast, these hormone results differ from those using the PMSG/hCG-stimulated ovary, suggesting that such ovaries may not be a completely valid model for ovarian steroid hormone metabolism in the normally cycling sow.

Is changing hypothalamic activity important for control of ovulation?

The activity of the hypothalamic gonadotrophin releasing hormone pulse generator in women with regular ovulatory and anovulatory menstrual cycles was assessed to see whether changes therein are important determinants of normal and impaired ovarian function. Endogenous gonadotrophin releasing hormone secretion was inferred by measurement of the pituitary luteinising hormone response by characterisation of pulsatile luteinising hormone release over eight hours on three occasions during the course of follicular development and once during the luteal stage of the same cycles. In 13 ovulatory cycles (serum progesterone concentration greater than 25 nmol/l) confirmed by ovarian ultrasonography a pronounced variability in luteinising hormone pulse patterns among subjects was compatible with ovulation. In the luteal stage of ovulatory cycles the luteinising hormone interpeak interval (85 min, range 42-125) was significantly longer than that during the early follicular (64 min, 40-103), mid-follicular (62 min, 37-107), and late follicular (59 min, 39-80) stages of the same cycles. Thus in ovulatory cycles no increase in frequency of the gonadotrophin releasing hormone pulse generator was detected during follicular development, though this activity decreased in the luteal stage. In five late follicular stage studies in which part of the preovulatory luteinising hormone surge was captured no change in pulse frequency of luteinising hormone was detected compared with the mid-follicular stage of the same cycles or when compared with the late follicular stage of other cycles when no luteinising hormone surge was captured. Though mean luteinising hormone concentrations in luteinising hormone surge series (36 IU/l) were high, the amplitude of luteinising hormone pulses (165%) was only slightly greater than during non-surge late follicular stage studies (145%). Hence no change in hypothalamic gonadotrophin releasing hormone activity is required to generate the preovulatory discharge of luteinising hormone in man, which occurs as a result of the sensitising action of rising oestradiol concentrations on pituitary responsiveness to the same hypothalamic input signal. Luteinising hormone pulse frequency, peak amplitude, and mean serum luteinising hormone concentrations in seven anovulatory cycles (progesterone concentration less than 10 nmol/l) were not different from those at comparable stages of ovulatory cycles. These data suggest that the primary abnormality in this group of regularly menstruating anovulatory women lies in the ovary rather than in the hypothalamic control of the anterior pituitary.

The detection of oviduct-specific proteins in the baboon (Papio anubis).

Oviducts obtained from cycling baboons were first flushed with saline, then minced and cultured in the presence of L-[3H] leucine. The flushings, tissue culture media, and solubilized tissues were analyzed by electrophoresis for secretory products that were oviduct-specific. The fluorographs of one-dimensional 7.5% slab gels demonstrated that a macromolecule of Mr 130,000 was present in the tissue culture medium of oviducts obtained during the mid-to late follicular stages, and absent during the mid-to late luteal stages. Two-dimensional analysis revealed that the Mr 130,000 band consisted of one basic protein and two acidic proteins. The basic protein formed the major component of this band and was the protein either absent or greatly reduced in intensity during the luteal stage. A macromolecule with similar electrophoretic properties was present in silver-stained, two-dimensional gels of follicular stage flushings, and absent in both luteal stage flushings and follicular stage serum. Two other macromolecules (Mr approximately equal to 160,000 and 88,000) appeared to be secretory products that were oviduct-specific. While both were present at all stages of the menstrual cycle, the Mr 160,000 protein showed a midcycle intensification, and the Mr 88,000 protein showed a midluteal stage intensification. Thus, the synthetic ability of baboon oviduct tissue and macromolecular content of the oviduct lumen changed with the stage of the menstrual cycle. The major alteration was in the synthesis and secretion of the Mr 130,000 basic macro-molecule, which was the major protein present during the follicular stage of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of male presence or absence on the reproductive cycle of celebes black ape females (Macaca nigra)

The effect of male presence on female menstrual cycles was studied in a group of 13 adult female and 3 adult male Celebes black apes (Macaca nigra) living in an outdoor enclosure. When males were absent, all cycling females had longer durations of maximal perineal swelling. Overall cycle length also appeared to be longer when males were absent, but there was no statistically significant increase. The data suggest that male absence results in a longer late follicular stage, perhaps indicating a delay in ovulation. Whereas these findings do imply that male presence may influence female macaque reproductive condition, it is acknowledged that this effect is much weaker than the demonstrated influence of female macaques on male reproductive state.

Cortisol, 17 alpha-OH, progesterone and androgen responses to a standardized ACTH-stimulation in different stages of the normal menstrual cycle.

The release of cortisol, 17 alpha-OH-progesterone, androstenedione and testosterone during a standardized ACTH-stimulation test was investigated in three different stage of the normal menstrual cycle, to conclude if there is any stage dependency on the release of these hormones. No statistically significant differences were observed between the three stages concerning cortisol and testosterone increase. The increase of androstenedione in the pre-ovulatory stage was significantly high than that seen during the early follicular phase of the cycle. The increase of 17 alpha-OH-progesterone in the luteal phase was significantly less than that of both the early and late follicular stages of the cycle. Progesterone levels showed a small, but significant increase after ACTH-stimulation, in both the early and late stage of the follicular phase. However, the levels remained within the normal range of the follicular phase. In the luteal phase no increase was seen after ACTH-stimulation. Oestradiol-17 beta levels did not change at all after ACTH stimulation. The stage dependency of androstenedione and 17 alpha-OH-progesterone is discussed. The described stage-dependency different increase of 17 alpha-OH-progesterone release can be of importance when the results of ACTH-tests are evaluated to detect carriers of congenital adrenal hyperplasia.

Transmission of circulating IgG into preovulatory mouse eggs

Mice were injected intravenously with radioactively labelled rat IgG or human serum albumin (controls) and their ovaries subsequently investigated by autoradiography at the light and electron microscopical levels. Both of the administered proteins reached the cytoplasm and nuclei of the oocytes at all except the late secondary and early tertiary follicular stages. The entry of the IgG into the eggs was higher than the entry of the albumin.

Cyclic changes in ovarian morphology during the menstrual cycle in Macaca mulatta.

AbstractTwenty‐seven mature female macaques were ovariectomized at various times during the menstrual cycle. The ovaries were sectioned serially and each vesicular follicle was measured and classified according to size and developmental state. Also noted was the presence of differentiated and undifferentiated glandular tissue.The data showed that when the corpus luteum was active the largest follicle in each animal was not less than 1 mm in diameter, and that when the corpus luteum began to degenerate follicular enlargement commenced. Most follicles became atretic when under 1 mm in diameter, except for a few in the late follicular stage. The maximal diameter for an atretic follicle was 3.1 mm and for a developing follicle 6.3 mm.Interstitial gland tissue (theca interna of atretic follicles) was noted in large amounts just before ovulation while minimal amounts were present throughout the rest of the cycle.Other glandular types occasionally seen were aberrant and accessory corpora lutea, cords of cells in the stroma and adrenal rests.