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The aim of this study in sheep ovaries was to determine the total number of granulosa cells in primordial follicles and at subsequent stages of growth to early antrum formation. The second aim was to examine the interrelationships among the total number of granulosa cells in the follicles, the number of granulosa cells in the section through the oocyte nucleolus, and the diameter of the oocyte. A third aim was to examine whether proliferating cell nuclear antigen labelling occurred in flattened granulosa cells in primordial follicles or was confined to follicles containing cuboidal granulosa cells. The follicles were classified using the section through the oocyte nucleolus by the configuration of granulosa cells around the oocyte as type 1 (primordial), type la (transitory), type 2 (primary), type 3 (small preantral), type 4 (large preantral), and type 5 (small antral). In type 1 follicles, the number of granulosa cells and oocyte diameter were highly variable in both fetal and adult ovaries. Each type of follicle was significantly different from the others (all P < 0.05) with respect to oocyte diameter, number of granulosa cells in the section through the oocyte nucleolus and total number of granulosa cells. Follicles classified as type 2, 3, 4 or 5 each corresponded to two doublings of the total granulosa cell population. The relationships between oocyte diameter and the number of granulosa cells (that is, in the section through the oocyte nucleolus or total population per follicle) could all be described by the regression equation loge X = a + b loge Y with the correlation coefficients R always > 0.93. For each pair of variables the slopes (b) for each type of follicle were not different from the overall slope for all types of follicle pooled. Immunostaining for proliferating cell nuclear antigen was observed in granulosa cells in type 1 follicles, as well as in the other types of follicle. These findings indicate that 'flattened' granulosa cells in type 1 follicles express an essential nuclear protein involved in cell proliferation before assuming the cuboidal shape. Thus, when considering factors that regulate specific phases of early follicular growth, it is important to consider: (i) the follicle classification system used; (ii) the animal model studied; (iii) whether type 1 follicles are all quiescent; and (iv) the likelihood that each follicle type represents more than one doubling of the population of granulosa cells.
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The aim of the present study was to establish whether the steroids, progesterone, androstenedione, testosterone and oestradiol, were present in the mesonephric–gonadal complex of female and male sheep fetuses around sexual differentiation (that is, from day 28 to day 45 of gestation, with sexual differentiation occurring at approximately day 32). A second aim was to test whether the mesonephric–gonadal complex, mesonephros (days 35–45 only) and gonad (days 35–45 only) were capable of steroid synthesis in vitro. The steroid contents in the mesonephric–gonadal complex were not detectable before sexual differentiation. However, from day 35 of gestation onwards, the mesonephric–ovarian complex contained mainly oestradiol and the mesonephric–testicular complex contained mainly testosterone: from day 35 until day 45 the increase in content of these two steroids exceeded the increase in the mass of tissue by more than fivefold. From day 40 to day 45 of gestation, the contents of the other steroids in the pathways to oestradiol increased progressively in both sexes but more in parallel with the increase in tissue mass. In contrast to the steroid contents in the tissue at recovery, the mesonephric–gonadal tissue from both sexes in tissue culture was able to synthesize most steroids before and after sexual differentiation and also to metabolise supplementary androstenedione to oestradiol. These findings suggest that many, if not all, of the steroidogenic enzymes in the pathway from cholesterol to oestradiol are present before sexual differentiation. Most of the aforementioned steroids were present in detectable amounts in isolated mesonephros and gonad of both sexes after sexual differentiation. Moreover, for both the isolated mesonephros and gonad, there were increases in the mean contents of most steroids after culture relative to the contents in the tissues at recovery. These data suggest that the mesonephros, as well as the gonad, in both sexes is capable of synthesizing steroid. It is concluded that, in the sheep fetus, the female and male gonads are steroidogenically active after sexual differentiation, that the steroidogenic enzymes develop before sexual differentiation, and that the mesonephros is a site of steroid synthesis.
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In female fetuses the Booroola gene (FecBB) is known to affect germ cell development and consequently the pattern of ovarian follicular growth during fetal and post-natal life. However in males, the role of this gene during fetal development is unknown. The aims of the study reported here were to examine the effects of the FecBB gene on development of male fetuses with respect to body and organ mass (for example, pituitary gland, adrenal and mesonephros), testes development, including numbers of germ cells, and also the plasma concentrations or tissue contents of the reproductive hormones (FSH, LH and testosterone) at days 40, 55, 75, 90 and 135 of gestation. The FecBB gene was found to influence litter size, bodymass, crown–rump length and testis mass at most stages of gestation. Some effects were also noted on the mesonephros at days 40 and 55 and on the pituitary and adrenal at days 90 or 135 of gestation. However, the FecBB gene was not observed to have an effect on the patterns of germ cell development, on pituitary content or plasma concentrations of immunoreactive or bioactive FSH or immunoreactive LH or testicular content of testosterone. When embryo transfer experiments were performed to eliminate the effects of litter size at days 40, 90 and 135 of gestation nearly all of the differences in bodymass, crown–rump length and organ mass disappeared. The only exception to this was at day 90 when bodymass continued to be lighter and crown–rump lengths smaller in the BB/B + fetuses compared with the ++ fetuses; the significance of this finding remains unknown. It is concluded that for Booroola male fetuses there are no direct effects of the FecBB gene on pituitary gonadotrophin function or testicular development after sexual differentiation. Moreover, although there may be temporal differences around day 90 of gestation, there are no long-term, direct effects of the FecBB gene on total body, adrenal, testis or pituitary mass. Collectively these findings for the male are similar to those for female fetuses except with regard to germ cell development.
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Summary. Bromocriptine administration in the form of slow-release injections to male blue foxes during March–May abolished the normal spring rise in plasma prolactin concentrations seen in May and June. The spring moult was prevented and the treated animals retained a winter coat of varied quality and maturity until the end of the study in August.
Plasma testosterone concentrations fell normally from March until August. Testicular regression was, however, delayed, although there were individual variations in response. Estimation by DNA flow cytometry in early July of the relative numbers of haploid, diploid and tetraploid cells in the testis showed that, in the treated animals, 74–80% of the cells were haploid (maturing germinal cells), 4–6% tetraploid (mainly primary spermatocytes) and the rest diploid cells (somatic cells and the remaining germinal cell types). In the control males, however, no haploid cells were detected and the majority of cells were diploid (93–99%). At castration in August, histological examination revealed various stages of testicular regression in the treated and control animals.
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Extensive extracellular matrix remodelling occurs within the lifespan of the corpus luteum, particularly during corpus luteum formation and regression. A major mechanism for the regulation of extracellular matrix remodelling is via local production of specific proteinase inhibitors, such as the serine proteinase inhibitor plasminogen activator inhibitor type-1 (PAI-1). The objective of the present study was to characterize the localization, ontogeny and regulation of PAI-1 expression within ovine corpora lutea. Urokinase binding activity was detected within medium conditioned by ovine luteal cells. Production of PAI-1 by ovine luteal cells was confirmed by immunoprecipitating it from labelled proteins in culture medium. mRNA encoding PAI-1 was present within developing (day 3), mature (day 10) and regressing (30 h after prostaglandin F2α injection on day 10 after the onset of oestrus) corpora lutea as demonstrated by in situ hybridization. The ontogeny of PAI-1 mRNA expression was characterized within corpora lutea collected on days 3, 7, 10, 13 and 16 after the onset of oestrus (n = 4, 4, 4, 3 and 4, respectively). Expression of PAI-1 mRNA did not differ during the luteal phase (P= 0.06), although a trend for an increase in the amount of PAI-1 mRNA was observed on day 16. Expression of PAI-1 mRNA was also examined during luteal regression in corpora lutea collected 0, 6, 12, 24 and 36 h after injection of prostaglandin F2α on day 10 after the onset of oestrus (n = 4 at each time). Relative PAI-1 mRNA concentrations changed significantly during luteolysis induced by prostaglandin F2α (P = 0.0002). Administration of prostaglandin F2α resulted in a transient sevenfold increase in PAI-1 mRNA 6 h after injection (P = 0.0001) but by 12 h the amounts had returned to values similar to those detected on day 10. We conclude that PAI-1 is a major secretory product of ovine luteal cells and that a transient increase in PAI-1 mRNA occurs during luteolysis induced by prostaglandin F2α. PAI-1 probably plays a key local role in the control of extracellular proteolysis during the luteal phase.
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Maternal endometrial leukaemia inhibitory factor (LIF) is required for successful implantation in mice. Mice with homozygous deletions in this gene fail to support implantation. The localization of immunoreactive LIF and the concentration of the mRNA encoding human LIF in normal endometrium during the menstrual cycle were investigated. The amount of RNA was low or undetectable in the proliferative phase but increased by approximately six times in the mid- to late secretory phase. The protein can only be detected by immunocytochemistry in glandular epithelium in the mid- or late secretory phase. To investigate the possible target for the endometrial LIF, we undertook reverse transcription–PCR analysis of early human embryos to determine whether they contain the mRNA encoding the LIF receptor. This study indicated that at the time of implantation in humans, the maternal endometrium produces LIF and that the blastocyst expresses LIF receptor mRNA and therefore may be capable of responding to this signal.
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Summary. The size distribution of marmoset luteal cells was determined on Days 6, 14 and 20 after ovulation in non-pregnant cycles and in early pregnancy. Image analysis was used to estimate the cell diameter of dispersed cells prepared from the marmoset corpus luteum (CL). Steroidogenic cells showed a size distribution consistent with one population of cells. There was a significant increase in mean cell diameter (P < 0·05) from Day 6 to Day 14 in pregnant and non-pregnant animals with no further increase on Day 20. Micrographs of marmoset luteal tissue showed cells of > 10 μm containing the organelles typical of steroid-producing cells, and smaller non-steroidogenic cells surrounding the steroid-producing cells. On the basis of microscopy, there were no areas within the CL where cell composition was noticeably different. In contrast, micrographs of human luteal tissue showed two types of steroidogenic cell; most cells were similar to those in the marmoset CL but a smaller population of smaller cells could be distinguished around the periphery and along vascular septa. It is likely that these smaller and larger types of steroidogenic cells are of theca and granulosa cell origin respectively, the two cell populations differing in the degree of electron density and amount of rough endoplasmic reticulum. A distinguishing feature between marmoset and human luteal cells was the shape of the mitochondrian which were considerably rounder in marmoset luteal cells. The origin of steroidogenic cells in the marmoset CL is unclear, although in marmosets and man the luteal cell types display morphological characteristics distinct from the large and small luteal cells described for CL of the domestic ungulates.
Keywords: luteal cell; size distribution; morphology; marmoset monkey; man
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Summary. Mature beef cows were actively immunized pre partum (N = 5) or post partum (N = 10) against a PGF-2α–ovalbumin conjugate or against ovalbumin alone (control; N = 5). All cows in the control group exhibited first oestrous cycles which were of short duration (⩽ 12 days). Mean specific serum binding to [3H]PGF-2α in the control group was consistently < 1%. In the pre-partum PGF-2α-immunized cows, lifespan and progesterone secretion of the first corpus luteum formed post partum was maintained for > 39 days. Specific serum binding to [3H]PGF-2α in pre-partum and post-partum PGF-2α-immunized cows was elevated. Lifespan of the first corpus luteum formed in post-partum PGF-2α-immunized cows was short (<10 days; N = 1), normal (mean = 22 days; N = 4) or maintained (>31 days; N = 5). Luteal lifespan was dependent upon serum PGF-2α antibody titres, with cows exhibiting higher titres frequently having prolonged luteal lifespans after first ovulation. We conclude that active immunization of beef cows against PGF-2α extends the lifespan and progesterone secretion of corpora lutea anticipated to be short-lived. These results support the concept that the shorter lifespan of some corpora lutea in post-partum cows is due to a premature release of PGF-2α from the uterus.
Keywords: active immunization; PGF-2α; corpus luteum; cattle
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Summary. Blood flow in the middle uterine artery was measured with electromagnetic blood flow probes, and placental lactogen in jugular and uterine venous plasma was estimated as total lactogenic activity using a radioreceptor assay. There was no circadian variation in uterine arterial blood flow in late pregnancy (Days 105– 124) and the pattern of blood flow varied between goats. Blood flow was quite stable for periods of up to 40 min although at other times a rapid fall (by up to 90%) was followed by a gradual recovery. These spontaneous changes lasting up to 30 min could not be consistently related to postural or behavioural changes. Acute decreases of about the same duration could also be induced by administration of adrenaline. In the short-term there was no association between uterine blood flow and total lactogenic activity in the peripheral circulation during spontaneous or adrenaline-induced depression of blood flow. More limited short-term observations on total lactogenic activity in the uterine vein also failed to show a relationship with blood flow in the uterine artery.
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The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.