The role of relaxin and oestrogen in the remodelling of connective tissue was investigated by testing collagenase-dispersed cells (3 × 105 cells per well) from the uterine cervix of gilts for relaxin binding and collagen secretion. Relaxin-binding sites on these cells were quantified by specific binding of a saturating dose of 125I-labelled monotyrosyl relaxin at optimal conditions. Oestrogen at doses from 0.4 to 50 ng ml−1 increased relaxin binding in a time- and dose-dependent manner. Scatchard plot analysis exhibited curvilinearity, which suggested two classes of relaxin-binding sites. The addition of relaxin (0, 100, 500 ng ml−1) alone (P < 0.05) or in combination with oestrogen (oestradiol benzoate: 0, 50, 250 ng ml−1) increased protein secretion into the culture medium. Hydroxyproline concentration (as an index of collagen) in the medium was increased (P < 0.05) only in the presence of both relaxin and oestrogen. Actinomycin D (500 ng ml−1) and cycloheximide (500 ng ml−1) inhibited hydroxyproline secretion induced by combined relaxin and oestrogen treatment. Dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl cAMP: 0, 0.1, 1.0, 5.0 mmol l−1) was a potent stimulator of hydroxyproline secretion. These results indicate that relaxin, probably via a cAMP pathway, stimulates hydroxyproline secretion in the presence of oestrogen through a protein- and RNA-synthesis dependent process. Oestrogen plays a role in augmenting the sensitivity of uterine cervical cells to relaxin in the pig.
C. Huang, Y. Li and L. L. Anderson
K. Tanaka, Z. D. Li and Y. Ataka
Summary. A system was developed for the in-vitro perfusion of the fowl ovary. The ovaries were isolated 16–18 h before expected ovulation of the first follicle of a clutch sequence and perfused at 41°C with Eagle's culture medium containing L-thyroxine and insulin. The efferent perfusion pressure was maintained at 30–40 mmHg. This model was used to investigate the mechanism of ovulation.
Addition of LH (1 U) to the perfusate induced ovulation (46%) but LH (1 U) + FSH (1 U) was more effective (88%; P < 0·05). Progesterone at 100 γg alone also induced ovulation (80%). Clomiphene prevented gonadotrophin-induced ovulation. These results suggest that progesterone may act directly on the ovary as a final hormone to induce ovulation in the domestic fowl.
Y Li, Michelle K Y Seah and C O'Neill
Reprogramming epigenetic modifications to cytosine is required for normal embryo development. We used improved immunolocalization techniques to simultaneously map global changes in the levels of 5′-methylcytosine (5meC) and 5′-hydroxymethylcytosine (5hmC) in each cell of the embryo from fertilization through the first rounds of cellular differentiation. The male and female pronuclei of the zygote showed similar staining levels, and these remained elevated over the next three cell cycles. The inner cells of the morula showed a progressive reduction in global levels of both 5meC and 5hmC and further losses occurred in the pluripotent inner cell mass (ICM) of the blastocyst. This was accompanied by undetectable levels of DNA methyltransferase of each class in the nuclei of the ICM, while DNA methyltransferase 3B was elevated in the hypermethylated nuclei of the trophectoderm (TE). Segregation of the ICM into hypoblast and epiblast was accompanied by increased levels in the hypoblast compared with the epiblast. Blastocyst outgrowth in vitro is a model for implantation and showed that a demethylated state persisted in the epiblast while the hypoblast had higher levels of both 5meC and 5hmC staining. The high levels of 5meC and 5hmC evident in the TE persisted in trophoblast and trophoblast giant cells after attachment of the blastocyst to the substratum in vitro. This study shows that global cytosine hypomethylation and hypohydroxymethylation accompanied the formation of the pluripotent ICM and this persisted into the epiblast after blastocyst outgrowth, and each differentiated lineage formed in the early embryo showed higher global levels of 5meC and 5hmC.
O. S. Gazal, Y. Li, C. Schwabe and L. L. Anderson
Pregnant ewes were injected with either the antiprogesterone, RU 486 (4 mg kg−1 body weight, i.m.; n = 5), 3000 iu relaxin (i.m.; n = 9), or diluent (n = 8) at 12:00 h on days 144 and 145, to determine its effect on progesterone and relaxin secretion, and on induction of lambing. RU 486 induced earlier lambing (P < 0.01) compared with diluent treatment, but relaxin treatment did not significantly reduce the interval to parturition. Mean injection–lambing intervals were 31 ± 2, 109 ± 23 and 121 ± 27 h for the RU 486, relaxin and diluent groups, respectively. There was no incidence of difficult birth (dystocia); all lambs were vigorous at birth; and placenta delivery was rapid (within 207 min) with RU 486 and relaxin treatments compared with diluent treated controls. Plasma progesterone concentrations averaged 11 ng ml−1 during the pretreatment period for all animals. RU 486 had a biphasic effect on progesterone concentrations, causing an initial increase (P < 0.05) within 2 h, and then an abrupt drop (P < 0.01) to 6 ng ml−1 by 18:00 h on day 145. Progesterone concentrations remained consistently lower (P < 0.05) in relaxin-treated ewes than in diluent-treated controls from days 144 to 147 and then began a steady decrease to 4 ng ml−1 on the day of parturition (days 149 and 150) in both groups. Immunoreactive relaxin concentrations in control ewes increased (P < 0.05) from 0.6 ng ml−1 to a peak of 3.9 ng ml−1 on day 146, but they were low (0.8 ng ml−1) at the time of parturition (day 150). RU 486 treatment abruptly increased (P < 0.05) circulating relaxin concentration, but the amplitude of this antepartum surge was greatly attenuated compared with that of diluent treated controls. Peak RU 486 concentrations in plasma were 7 and 9 ng ml−1 within 2 h after first and second i.m. injection of the hormone, respectively, and stabilized at 4 ng ml−1 at the time of induced lambing (day 145). The results reveal that an antepartum relaxin surge occurs in sheep 4 days before normal parturition (day 150), but that RU 486 greatly attenuates the relaxin surge and abruptly decreases circulating progesterone concentration with an induced parturition (day 145). The results indicate that RU 486 can precisely control the time of parturition in sheep in late pregnancy without detrimental effects of dystocia, retention of placenta or delayed postpartum fertility.
C Rollo, Y Li, X L Jin and C O’Neill
Acetylation of histone proteins is a major determinant of chromatin structure and function. Fertilisation triggers a round of chromatin remodelling that prepares the genome for the first round of transcription from the new embryonic genome. In this study we confirm that fertilisation leads to a marked progressive increase in the level of histone 3 lysine 9 acetylation in both the paternally and maternally derived genomes. The culture of zygotes in simple defined media caused a marked increase in the global level of acetylation and this affected the male pronucleus more than the female. The culture created a marked asymmetry in staining between the two pronuclei that was not readily detected in zygotes collected directly from the reproductive tract and was ameliorated to some extent by optimized culture media. The increased acetylation caused by culture resulted in increased transcription of Hspa1b, a marker of embryonic genome activation. Pharmacological analyses showed the hyperacetylation of H3K9 and the increased expression of Hspa1b caused by culture were due to the altered net activity of a range of histone acetylases and deacetylases. The marked hyperacetylation of histone 3 lysine 9 caused by culture of zygotes may serve as an early biomarker for the effects of culture on the normal function of the embryo. The results also provide further evidence for an effect of the stresses associated with assisted reproductive technologies on the normal patterns of epigenetic reprogramming in the early embryo.
Xihe Li, Y Qin, Sandra Wilsher and W R Allen
Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-α-tubulin antibody for microtubules and a rabbit anti-γ-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of γ-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of γ-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.
B. L. Song, D. R. Peng, H. Y. Li, G. H. Zhang, J. Zhang, K. L. Li and Y. Q. Zhao
Summary. The inhibition of the proteolytic activity of acrosin in human spermatozoa by butyl p-hydroxybenzoate was assessed by the gelatin substrate film method. Compared with a typical acrosin inhibitor, TLCK, the inhibitory activity of butyl p-hydroxybenzoate to acrosin was much more effective (20 times) than that of TLCK, proving that butyl p-hydroxybenzoate was a potent acrosin inhibitor. The effect of butyl p-hydroxybenzoate on membrane function of human spermatozoa was evaluated using a sperm-tail hypoosmotic swelling test and supravital stain method. A good correlation (r = 0·92) was observed between the % spermatozoa with normal membrane function and the % live spermatozoa after treatment of the spermatozoa with butyl p-hydroxybenzoate for 1 min, indicating that the death of spermatozoa caused by butyl p-hydroxybenzoate is probably due to impairment of sperm membrane function. Both the inhibitory effect on acrosin and the adverse effect on membrane function suggest that butyl p-hydroxybenzoate could be developed as a new vaginal contraceptive.
Keywords: butyl p-hydroxybenzoate; proteinase inhibitor; sperm; acrosin; membrane; man
C. J. Huang, Y. Li, M. H. Stromer and L. L. Anderson
Summary. Insulin-like growth factor I (IGF-I) is involved in paracrine/autocrine regulation of gonadal steroidogenesis and peptide hormone biosynthesis. This study was designed to determine whether IGF-I alone, or an interaction of IGF-I, is involved in augmenting the actions of luteinizing hormone (LH) and prolactin in controlling relaxin and progesterone secretion from ageing corpora lutea of hysterectomized gilts at days 110, 113 and 116 after oestrus. Luteal tissue slices were incubated for 8 h with IGF-I (0, 50, 300 ng ml−1), LH (0, 100, 1000 ng ml−1), and prolactin (0, 100, 1000 ng ml−1) alone or in combination. Progesterone and relaxin concentrations were determined by radioimmunoassay of spent medium and of homogenates from luteal tissue slices before and after incubation. Porcine luteal tissue from day 110 had a net output of 25 ng progesterone and 26 ng relaxin in the control and of 65 ng progesterone and 2125 ng relaxin in the combined IGF-I, LH and prolactin treatment mg−1 of luteal tissue, respectively. IGF-I, LH and prolactin alone or in combination significantly increased (P<0·01) progesterone production by luteal tissue from day 110, but they were partially effective at day 113 and ineffective at day 116. By contrast, the same hormone treatments increased relaxin production by luteal tissue from days 110 and 113. Even at day 116, prolactin alone or with LH or IGF-I continued to stimulate relaxin production. In conclusion, IGF-I augments the ability of prolactin and LH to increase relaxin production by ageing corpora lutea; however, a decrease in progesterone secretion and an increase in relaxin secretion at day 113 indicate that different mechanisms control progesterone and relaxin secretion in pigs.
Keywords: IGF-I; gonadotrophins; relaxin; progesterone; ageing luteal cells; pig
C L Lu, J Yan, X Zhi, X Xia, T R Wang, L Y Yan, Y Yu, T Ding, J M Gao, R Li and J Qiao
Fertility preservation is an important type of frontier scientific research in the field of reproductive health. The culture of ovarian cortices to i) initiate primordial follicle growth and ii) procure developing follicles for later oocyte maturation is a promising fertility preservation strategy, especially for older women or cancer patients. At present, this goal remains largely unsubstantiated in primates because of the difficulty in attaining relatively large follicles via ovarian cortex culture. To overcome this hurdle, we cultured macaque monkey ovarian cortices with FSH, kit ligand (KL), basic fibroblast growth factor (bFGF), and/or epidermal growth factor (EGF). The various factors and factor combinations promoted primordial follicle development to different extents. Notably, both bFF (bFGF, 100 ng/ml and FSH, 50 ng/ml) and KF (KL, 100 ng/ml and FSH, 50 ng/ml) contributed to the activation of primordial follicles at day 12 (D12) of culture, whereas at D18, the proportions of developing follicles were significantly higher in the bFF and KF groups relative to the other treatment groups, particularly in the bFF group. Estradiol and progesterone production were also highest in the bFF group, and primary follicle diameters were the largest. Up until D24, the bFF group still exhibited the highest proportion of developing follicles. In conclusion, the bFGF–FSH combination promotes nonhuman primate primordial follicle development in vitro, with the optimal experimental window within 18 days. These results provide evidence for the future success of human ovarian cortex culture and the eventual acquisition of mature human follicles or oocytes for fertility restoration.
D. A. Redmer, Y. Dai, J. Li, D. S. Charnock-Jones, S. K. Smith, L. P. Reynolds and R. M. Moor
The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2–4), mid- (day 8) and late (days 14–15) stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF VEGF120, VEGF164, and VEGF188) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2–4 than on day 8 or days 14–15. Densitometric analysis of gels from the RNase protection assay showed that VEGF120 represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.