Regeneration of atretic sheep ovarian follicles in vitro.

Large (4--6 mm diam.) and small (2--3 mm) atretic follicles were removed from sheep ovaries during the luteal phase of the cycle and maintained in organ culture without hormonal supplementation for up to 5 days. The structure, cell dynamics and steroid-producing capacity of the follicles were compared with those of non-atretic follicles of similar size. The granulosa layer of the atretic follicles invariably regenerated in culture, increasing in thickness more than 2- and 4-fold in large and small follicles respectively. This could not be accounted for by cell division which remained low throughout the culture period. In contrast, non-atretic follicles showed high mitotic activity during the first 24 h in culture: this was not associated with an increase in granulosa thickness in large follicles although there was a 4-fold increase in small ones. An increase in internuclear spacing, a measure of cell size plus intercellular space, partly accounted for the increase in granulosa thickness in atretic follicles. Even when granulosa cells remained in close apposition there was an almost total absence of gap junctions, a prominent feature in the granulosa of non-atretic follicles both in vivo and in vitro. Pyknotic nuclei and atretic bodies rapidly disappeared from the regenerating granulosa layer. The theca interna was restored in culture to a state ultrastructurally closely resembling that of non-atretic follicles in vivo. Total steroid secretion (oestradiol-17beta, testosterone plus progesterone) into the culture medium (pmol.mg tissue-1.24 h-1) was the same for atretic and non-atretic follicles of comparable size. There was, however, a marked difference in the type of steroid produced, largely related to a loss of aromatizing capacity in atretic follicles. The predominant steroid secreted by large non-atretic follicles was oestrogen, with slightly smaller amounts of testosterone, whereas the principal steroid secreted by large atretic follicles was progesterone. In small non-atretic and atretic follicles, the predominant steroid was testosterone, but the non-atretic follicles also secreted appreciable amounts of oestrogen. Addition of FSH to the culture medium did not restore aromatizing capacity to the atretic follicles.     

Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrus cycle

Eight hundred and seven bovine antral follicles from 2 mm to 20 mm in diameter were dissected free of stromal tissue, measured, qualified and divided into 36 groups according to size, quality and stage of cycle. The follicular fluid was collected and assayed by RIA for oestradiol-17beta, testosterone and progesterone. The steroid hormone concentrations vary with follicular size, degree of atresia and stage of the cylce. Non-atretic follicles of less than 8 mm are generally androgen-dominated and non-atretic follicles of more than 11 mm are oestrogen-dominated. Follicles betwen 8 mm and 11 mm are intermediate in this respect. Degeneration leads to a gradual decrease of oestradiol-17beta and testosterone concentration and increase of progesterone. It is suggested that the ratio of oestradiol-17beta/testosterone and oestradiol- 17beta/progesterone and oestradiol-17beta/testosterone + progesterone cannot generally be used to discriminate between non-atretic and atretic follicles. Large follicles present during the early luteal stage contain as much oestradiol-17beta in the follicular fluid as large follicles during the follicular stage, whereas large follicles of the luteal stage contain only 15% of the maximal amount of the latter's. This and other presented data support the statement that follicles present during the early luteal, late luteal and follicular stages of the cycle belong to different groups of growing follicles. It has been concluded that groups of macroscopically qualified follicles can be distinguished from each other by the steroid hormone concentration in the follicular fluid. It is therefore possible to predict the hormonal environment of the oocyte in any individual follicle of a defined size and quality.     

Inhibin production in vitro by granulosa cells from Booroola ewes which were either homozygous or non-carriers of a fecundity gene influencing their ovulation rate

The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10-13 of the oestrous cycle, from Booroola x Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3-4.5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P less than 0.05) than that by cells from non-atretic 3-4.5 mm diameter ++ follicles, but similar to that of cells from non-atretic greater than or equal to 5 mm diameter ++ follicles. The production of oestradiol-17 beta by cells cultured in the presence of testosterone (1 microgram/ml) followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17 beta production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17 beta synthesis, testosterone generally had a slight, stimulatory effect on inhibin production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between aromatase activity, follicular fluid oestradiol-17 beta and testosterone concentrations, and diameter and atresia of individual ovine follicles

Aromatase activity was measured in granulosa cells using a 1-h in-vitro assay. This activity correlated with the concentration of oestradiol-17 beta and the ratio of oestradiol-17 beta to testosterone in follicular fluid of individual follicles ranging from 1.5 to 7.0 mm diameter. These data show an 8-10-fold difference in aromatase activity between small and large follicles and that aromatase activity per cell increased in small non-atretic follicles (less than 3.5 mm) whereas it remained relatively constant in large nonatretic follicles (greater than or equal to 3.5 mm). Aromatase activity was much lower in follicles at more advanced stages of atresia. Atresia was assessed using the morphological and the morphometric methods (% of maximum number of granulosa cells/follicle). Although the morphological method of assessment was preferable to the morphometric method, it did not differentiate a decrease in aromatase activity as a very early event in the atretic process. We believe this is due to the inability of these methods to detect follicles in the initial stages of atresia.     

Initiation of follicular maturation during mid-pregnancy by the administration of LH in the rat

Pregnant rats were injected twice daily for 1-3 days (Days 13-16 of pregnancy) with various doses of ovine LH. Follicular maturation was determined by the ability of the follicles to ovulate in response to 10 i.u. hCG as well as by endogenous production of oestradiol-17 beta and inhibin. In control animals, no ovulation was induced by hCG given on Day 16 of pregnancy. An injection of hCG on Day 16 of pregnancy, however, induced ovulation in LH-treated animals (6.25-50.0 micrograms LH per injection, s.c. at 12-h intervals from Days 13 to 16). Concentrations of oestradiol-17 beta and inhibin activity in ovarian venous plasma increased after the administration of LH, indicating that development of ovulatory follicles had been induced. Abolishing the decline in plasma LH values therefore induced maturation of a new set of follicles or prevented the atresia of large antral follicles usually seen at this time of pregnancy. Plasma and pituitary concentrations of FSH decreased in LH-treated animals compared with those in control animals. Concentrations of progesterone, testosterone and oestradiol-17 beta in the peripheral plasma were not significantly different between the two groups. These results suggest that the increase in inhibin secretion from the ovary containing maturing follicles after LH treatment may suppress the secretion of FSH from the pituitary gland. These findings indicate that (1) the development of ovulatory follicles can be induced by the administration of exogenous LH during mid-pregnancy in the rat and (2) basal concentrations of FSH are enough to initiate follicular maturation even in the presence of active corpora lutea of pregnancy, when appropriate amounts of plasma LH are present.     

Ovarian follicular activity in Booroola lambs with and without a fecundity gene

The ovaries of 3-month-old Booroola lambs which were heterozygous carriers of a major gene (F) influencing the ovulation rate in mature ewes (i.e. F + lambs) were compared to those ofsimilarly-aged Booroola lambs which were non-carriers of the F-gene (i.e. ++ lambs). The ovaries of the F + Booroola lambs were significantly lighter (P less than 0.01) than those of ++ lambs even though the mean +/- s.e.m. number of follicles (greater than or equal to 1 mm diam.) in the F + lambs was greater than that in the ++ lambs (i.e. F + lambs, 30.2 +/- 2.5 follicles; ++ lambs, 18.4 +/- 1.2 follicles; P less than 0.01). In granulosa cells from non-atretic follicles (greater than or equal to 1 mm diam.) from F + and ++ Booroola lambs, FSH (NIAMDD-FSH-S16) doses of 100 and 1000 ng/ml caused significant stepwise increases (P less than 0.05) in cyclic adenosine 3',5'-monophosphate (cAMP) production compared to that achieved at FSH doses of 0 and 1 ng/ml or at any FSH dose in cells from atretic follicles. However, no significant differences in FSH-induced cAMP production were noted with regard to Booroola genotype or follicular diameter. None of the granulosa cell preparations from non-atretic follicles of 1-2.5 mm diameter from F + lambs (N = 13) or from non-atretic follicles of 1-4.5 mm diameter from ++ lambs (N = 16) responded to LH (NIAMDD-LH-S24; 10 or 1000 ng/ml) to produce significantly more cAMP than did the controls. In contrast, the granulosa cell preparations from non-atretic follicles of 3-4.5 mm diameter from F + lambs (N = 4) and from non-atretic follicles of greater than or equal to 5 mm diameter of ++ lambs (N = 4) produced significantly more cAMP (P less than 0.05) in response to LH (1000 and/or 10 ng/ml) relative to that in the controls. The theca interna from follicles of lambs of both genotypes had functional LH receptors as judged by the androstenedione responses to exogenous LH although no genotypic differences were noted. In F + lambs, the follicular fluid concentrations of testosterone but not oestradiol (i.e. in 1-4.5 mm diam. follicles) and granulosa cell aromatase activity (i.e. in 3-3.5 mm diam. follicles) were significantly higher (both P less than 0.05) than in corresponding follicles or cells from ++ lambs. Collectively the results suggest that the Booroola F-gene influences the composition and function of sheep ovaries before puberty.     

Inhibitory effect of progesterone on cell death of mouse uterine epithelium

The protective effect of progesterone against cell death of mouse uterine epithelium was evaluated by examining the retention of 5'-[125I]iodo-2'-deoxyuridine [( 125I]IdUrd) incorporated into the whole uterus and the apoptotic index (percentage of apoptotic cells in total cells), which is a good index of physiological cell death. Castrated adult female mice were given a daily injection of oestradiol-17 beta for 3 days, and then an injection of [125I]IdUrd. They were then divided into 4 groups, which received a daily injection of vehicle only, oestradiol-17 beta (E), progesterone (P), or both oestradiol-17 beta and progesterone (EP), and were killed at intervals during these treatments for determination of 125I radioactivity retained in the whole uterus. On treatment with vehicle only, the 125I radioactivity retained in the uterus decreased rapidly, but treatment with E, P or EP reduced the loss of 125I radioactivity significantly. Progesterone did not antagonize the effect of oestradiol-17 beta on the 125I radioactivity retained in the uterus. The apoptotic index of uterine cells was examined by a similar experimental protocol, but without injection of [125I]IdUrd. In the group treated with vehicle only, the apoptotic indices of both luminal and glandular epithelia increased markedly, but the injection of E, P or EP suppressed these increases significantly. Progesterone did not antagonize the effect of oestradiol-17 beta on the apoptotic index. The apoptotic index of stroma was not affected by the injection of E, P or EP. On the other hand, progesterone completely inhibited the increase in the mitotic index of uterine epithelia induced by oestradiol-17 beta. These results show that progesterone alone or in combination with oestrogen reduced cell death in mouse uterine epithelium and that the effects of oestrogen and progesterone on uterine cell death were independent of their actions on cell division.     

A comparison of histological and non-histological indices of atresia and follicular function

Histological indices of atresia for bovine follicles greater than or equal to 5 mm in diameter were compared with potential non-histological indices of atresia such as opaqueness of the exposed surface of non-excised follicles, concentrations of steroids in follicular fluid (FF) and specific binding of gonadotropins by granulosal cells. Each non-excised follicle was classified as clear (n=86), intermediate (n=79), or opaque (n=115), on the basis of the appearance of its exposed surface. A section of tissue from each follicle was evaluated histologically for atresia and assigned to one of the following categories: non-atretic, intermediately atretic, strongly atretic, or luteinized-atretic. Concentrations of estradiol (E), progesterone (P), and testosterone (T) and capacity of granulosal cells to bind radioactive ovine follicle-stimulating hormone (oFSH) and human chorionic gonadotropin (hCG) were determined for each follicle. Overall incidence of atresia was similar for clear (n=66%), intermediate (60%), and opaque (72%) follicles. Opaque follicles, however, were more likely to be strongly atretic (42%) than were clear (21%) or intermediate (23%) follicles. Non-atretic and intermediately atretic follicles had similar concentrations of E, P, and T and similar capacities to bind gonadotropins. Strongly atretic and luteinized-atretic follicles contained a higher concentration of P, lower E, and a reduced capacity of granulosal cells to bind oFSH than non-atretic and intermediately atretic follicles. A ratio of P:E in FF greater than or equal to 10 usually (greater than 90%) indicated that a follicle was atretic. However, lesser ratios of P:E did not accurately indicate whether follicles were atretic.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.     

Endocrine changes during pregnancy, parturition and the early post-partum period in the llama (Lama glama)

Mean (+/- s.d.) pregnancy length for the 14 llamas in this study was 350 +/- 4.5 days. Plasma progesterone concentrations increased by 5 days after mating and remained elevated (greater than 2.0 ng/ml) throughout most of pregnancy. At about 2 weeks before parturition, plasma progesterone concentrations began to decline, dropped markedly during the final 24 h before parturition, and returned to basal concentrations (less than 0.5 ng/ml) by the day of parturition. The combined oestrone + oestradiol-17 beta and oestradiol-17 beta concentrations varied between 6 and 274 pg/ml and 4 and 114 pg/ml, respectively, during the first 9 months of pregnancy. Concentrations increased between 9 months after mating and the end of pregnancy with peak mean concentrations of 827 +/- 58 (s.e.m.) pg oestrone + oestradiol-17 beta/ml (range: 64-1658) and 196 +/- 10 pg oestradiol-17 beta/ml (31-294) during the last week of pregnancy. Concentrations then declined to 87 +/- 14 pg oestrone + oestradiol-17 beta/ml (7-488) and 25 +/- 5 pg oestradiol-17 beta/ml (2.5-142) during the first week post partum. Plasma cortisol concentrations varied between 2.6 and 51.9 ng/ml (14.0 +/- 0.5) from mating until 2 weeks before parturition when the concentrations began to decline. Only a slight increase in plasma cortisol concentrations was observed in association with parturition. Plasma triiodothyronine concentrations varied between 0.5 and 4.5 ng/ml (1.9 +/- 0.1) throughout pregnancy and the periparturient period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macroscopic classification of bovine follicles and its validation by micromorphological and steroid biochemical procedures

Bovine antral follicles ranging between 2 and 20 mm in diameter were dissected free of stromal tissue and classified under a stereomicroscope using criteria similar to those of Moor et al. (1978). For light-microscopic evaluation of the macroscopic classification, 191 follicles were fixed in Bouin's fluid, embedded in paraffin, sectioned through the cumulus and oocyte and stained with haematoxylin-eosin. To study the relation between the macroscopically determined morphological characteristics and the oestradiol-17 beta (E2) level in the follicular fluid (FF), 39 follicles of one animal were selected. The E2 level, expressed as pmol/ml of FF, was estimated by RIA. The overall correlations between macroscopic classification and histological qualification of non-atretic and atretic follicles were 93.3 and 95.5 p. 100, respectively. Furthermore, the results indicated that the E2 level of small non-atretic follicles was lower than that of larger follicles and that the level in the FF decreased with increasing degeneration. It is concluded that macroscopic criteria offer a highly reliable means of identifying bovine follicles in terms of both micromorphology and functionality.     

Profiles of oestradiol-17 beta and progesterone and follicular development during the reproductive season in mink (Mustela vison).

Plasma concentrations of oestradiol-17 beta and progesterone were studied in yearling mink females. The blood samples were collected from 2 March until 13 April in females not subjected to mating and in females mated on two consecutive days, early or late in the breeding season, or with 8-9 days between matings. Peaks in oestradiol-17 beta were recorded on the day of first mating, in relation to the second wave of growing follicles, and in early April, around the time when implantation should have occurred. Significant rises in progesterone were recorded from 17 to 21 March and were slightly later in females mated late in the season. Histological studies of ovaries from unmated females revealed that the number of 'active' follicles exceeded the number of degenerated or luteinized follicles until 7 April, after which the number of degenerated follicles increased rapidly. Degeneration was followed by luteinization. On 15 April, ovaries were collected from two females having 15 luteinized follicles each. These females had increased plasma concentrations of progesterone. These studies indicate that, in female mink, peaks in oestradiol-17 beta coincide with the first mating as a result of the copulatory act and that unmated females appear to experience a luteal phase in the absence of ovulation.     

Luteinization of bovine granulosa cells and corpus luteum formation associated with loss of androgen-aromatizing ability.

The relative aromatizing ability of bovine luteinizing granulosa cells and dispersed luteal cells in tissue culture was studied. Luteinization of granulosa cells, as indicated by steadily increasing progesterone production (from 50 to 300 ng/10(5) cells/day over 4--5 days), was accompanied by a dramatic reduction in their capacity to aromatize exogenous androgen; oestradiol-17 beta production falling from 200 to less than 10 ng/10(5) cells/day over 4--5 days. Luteal cells also had only a very limited capacity to aromatize exogenous androgen, maximum oestradiol-17 beta production being less than 600 pg/10(5) cells/day. The loss in aromatizing capacity of granulosa cells during luteinization was also reflected in the relative endogenous steroid content of non-luteinized granulosa cells and luteal tissue, the former containing high levels of oestradiol-17 beta, less than or equal to 28 ng/mg protein, while the latter, although containing substantial amounts of testosterone, less than or equal to 5.7 ng/g tissue, contained very little oestradiol-17 beta, less than or equal to 0.35 ngG TISSUE. These findings suggest that luteinization of bovine granulosa cells and subsequent corpus luteum formation is associated with a loss in androgen aromatase activity.     

Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH

Granulosa cells from fully differentiated bovine follicles were cultured in serum-free medium for 4 days. At the end of culture, the number of viable cells was low (10-15% of cells plated on day one) and only progesterone secretion responded to FSH. Insulin increased the number of viable cells at the end of culture (ED50 # 70 ng/ml) and stimulated progesterone secretion (ED50 # 50 ng/ml); the secretion of oestradiol-17 beta over basal value was evident only for concentrations of 1000 and 10,000 ng/ml. FSH acted synergistically with insulin to modify steroid secretion. In the presence of 50 ng/ml of insulin, dose-response studies indicated that secretion of progesterone was maximal at 10 ng/ml of FSH and plateaud thereafter, while oestradiol output peaked at 2 ng/ml of FSH, decreasing at higher concentrations. When cells were seeded in wells precoated with fibronectin, a comparison with cells cultured on plastic showed an increase (30-40%) in the number of viable cells at the end of culture and in oestradiol secretion but a decrease in progesterone output. These results indicate that granulosa cells from large bovine follicles, cultured in a serum-free medium containing insulin, maintain their steroidogenic potency for at least 4 days. Moreover, they show that oestradiol and progesterone synthesis are differentially sensitive to FSH concentrations and that fibronectin increases oestradiol secretion in response to FSH.     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.     

Effect of follicular fluid and oestradiol on the luteinization of rat granulosa cells in vitro.

The effect of pig follicular fluid (FF), total or oestrogen-free, and of oestradiol-17 beta on the luteinization and progesterone secretion of rat granulosa cells was investigated during 4 days in culture. Both FF and oestrogen-free FF modified the differentiation of the granulosa cells, particularly their size and the appearance of their nucleoli. Addition of total FF or oestradiol-17 beta (50, 100 or 500 ng/ml) to the control medium markedly increased the progesterone secretion of the granulosa cells, but oestrogen-free FF and dialysed fetal calf serum had no effect. It was concluded that (1) FF could modify the morphological changes of the rat granulosa cells in vitro, but could not inhibit their secretion of progesterone, (2) the granulosa cells were able to synthesize progesterone regardless of their stage of differentiation, (3) oestradiol 17 beta had a direct stimulatory effect on progesterone secretion by granulosa cells in vitro. The possible mode of action of FF upon luteinization is discussed.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of follicular atresia and size on the developmental competence of bovine oocytes: a study using the well-in-drop culture system

The effect of granulosa cell (GC) apoptosis and follicle size on the competence of bovine oocytes were studied using a well-in-drop (WID) oocyte/embryo culture system, which allows identification of follicular origin. Hatching rates of blastocysts did not differ (P>0.05) between oocytes cultured in the WID system (13%) and those cultured in the conventional group system (16%). Hatching rates of blastocysts were higher (P<0.05) in early atretic (17%) than in non-atretic (8%) and late atretic follicles (10%) of the same size (4-8mm), and in 6-8mm (22%) than in 4-5mm follicles (15%) at the early atretic stage. More oocytes (P<0.05) from late atretic (17%) than from non-atreteic (7%) or early atretic follicles (9%) of the same size (4-8mm) were arrested at Grade 1 cumulus expansion (only cells in the peripheral two layers began to expand). Similarly, more oocytes from 2 to 3mm follicles (30%) than from 6 to 8mm follicles (21%) at the same (late) atretic stage had Grade 1 cumulus expansion (P<0.05). Hatching blastocyst percentages of oocytes with Grade 3 (all layers of the cumulus except corona radiate cells expanded) or Grade 4 (full) cumulus expansion were higher in early atretic (20%) than in non-atretic (13%) or late atretic follicles (12%). Hatching blastocyst percentages of oocytes from follicles at the early atretic stage increased as cumulus expanded from Grade 2 (9%) to Grade 4 (27%). Regardless of the degree of follicle atresia, 72-76% of the floating cells in the follicular fluid (FF) were undergoing apoptosis. The floating cell density in FF was highly (r=0.6-0.7) correlated with oocyte developmental potency. In conclusion, the WID culture system was as efficient as group culture and allowed identification of follicular origin. Furthermore, the developmental potential of oocytes was affected by GC apoptosis, follicle size and cumulus expansion, and the floating cell density in FF could be used as a simple and non-invasive marker of oocyte quality.     

Interleukin-1 alpha increases thecal progesterone production of preovulatory follicles in cyclic hamsters

Adult cyclic hamsters were used to study the effects of interleukin-1 alpha (IL-1 alpha) on in vitro steroidogenesis in preovulatory follicles. IL-1 alpha increased progesterone secretion by preovulatory follicles during a 24-h incubation in RPMI-1640 medium containing hCG (100 mIU/ml) (progesterone levels: 17.5 +/- 2.2 vs. 10.6 +/- 1.9 ng/follicle/ml, p less than 0.05). IL-1 alpha alone had no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 +/- 1.0 vs. 5.8 +/- 0.4 ng/2 thecae/ml, p less than 0.01) and not the granulosa cells (6.6 +/- 0.2 vs. 6.8 +/- 0.5 ng/20,000 viable granulosa cells/ml). IL-1 alpha also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-1 alpha. IL-1 alpha at 5-50 U/ml maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-1 alpha was observed until the 12th hour of incubation. The effects of IL-1 alpha on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea were examined. IL-1 alpha in the presence of hCG also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lutea, however, IL-1 alpha did not alter steroidogenesis. These results indicate that IL-1 alpha stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrine changes, with special emphasis on oestradiol-17 beta, prolactin and oxytocin, before and during labour and delivery in goats

Jugular plasma concentrations of oestradiol-17 beta, prolactin, progesterone and 13,14-dihydro-15-keto-prostaglandin F-2 alpha (PGFM) were measured at 2-h intervals during the last 4 days of pregnancy in 6 goats. During advanced labour and delivery, samples were obtained more frequently and assayed for oxytocin. The animals were housed in a barn with continuous dim lighting. A distinct pattern of oscillation in prolactin concentrations, with peaks during the late afternoon, was apparent during the last 3 days. Geometric means of peak concentrations doubled each day and became of longer duration; night-time nadir values remained low except during the last night before parturition. A progressive increase in oestradiol-17 beta, with mean levels doubling every 36 h, was apparent during the last 3 days. There was no sharp pre-partum increase in oestradiol-17 beta. Correlated (r = 0.83) with the increase in oestradiol-17 beta was a gradual increase in PGFM and when the latter reached approximately 1000 pg/ml, the non-reversible decline in progesterone reflecting pre-partum luteolysis occurred. Subsequent changes in PGFM related closely to an approximately 20-fold increase in the ratio of oestradiol-17 beta to progesterone until maximal PGFM levels of 26.5 +/- 4.2 ng/ml were reached at delivery. Basal concentrations of oxytocin (8-15 microU/ml) were measured before the last 60 min and markedly higher, though erratic, concentrations were detected at various times before appearance of the allantochorion. Maximal oxytocin values (range 180-1570 microU/ml) occurred within minutes before or after delivery of the first fetus. The results suggest that increased pre-partum production of oestradiol-17 beta, in addition to provoking sufficient release of prostaglandins to cause luteolysis, may modulate either the sensitivity or set-points for an endogenous rhythm in prolactin secretion at the end of pregnancy. The nature of the oxytocin changes suggest that, after labour has evolved sufficiently, delivery is precipitated by an abrupt increase in oxytocin secretion.