Incidence Of Atresia Research Articles

Influence of diet on growth, condition and reproductive capacity in Newfoundland and Labrador cod (Gadus morhua): Insights from stable carbon isotopes (δ13C)

Abstract Cod populations in Newfoundland and Labrador waters have shown differing growth, condition and recruitment since near-universal declines in these properties during the cold period of the late 1980s and early 1990s. To assess the influence of variable prey communities on these parameters, we compared cod energetics and diet in populations off Labrador and the northeast and south coasts of Newfoundland. Many properties were highest in the southern group(s) and lowest in the northern group(s), including growth, somatic condition, liver index and age-at-maturity. Most differences could be explained by variations in diet, as measured by stomach contents and stable carbon isotopes (δ13C). The diet of Labrador cod consisted almost entirely of northern shrimp (Pandalus borealis), and these cod displayed the most benthic δ13C signatures. Northeast cod had a more varied diet that included capelin and other fish, but still had mostly benthic δ13C signatures, suggesting the importance of benthic prey like shrimp in this population. South coast cod exhibited the most varied diet, including capelin (Mallotus villosus), zooplankton, crabs and other fish, and had the most pelagic δ13C signatures. Among and within populations, the benefits of a more pelagic diet in medium-sized (30–69 cm) cod included higher somatic condition, higher liver index (lipid stores) and greater spawning potential (decreased incidence of atresia). It is hypothesized that major rebuilding of Newfoundland and Labrador cod stocks will require a return to a system that supports mostly pelagic feeding (i.e. capelin) in cod.

Determinate versus indeterminate fecundity in horse mackerel

No consensus has been reached yet on the question whether horse mackerel has a determinate or an indeterminate fecundity. This problem has been discussed based on three main criteria: (a) oocyte size frequency distribution, (b) the number of yolked oocytes in the ovary and (c) atresia. The analysis of the oocyte size frequency distribution showed that no distinct hiatus could be observed between pre-vitellogenic and vitellogenic oocytes. The number of yolked oocytes in the ovary was found higher in imminent spawning ovaries than in the pre-spawing, while a greater number of late yolked oocytes was found in recently spawning ovaries than in the pre-spawning. High incidence of atresia was also found in recently spawning (6–22%) and spent ovaries (over 29%). Those findings indicate that horse mackerel has most probably an indeterminate fecundity.

KIT LIGAND STIMULATES ACTIVATION OF BOVINE PRIMORDIAL FOLLICLES IN VITRO

The regulation of activation of primordial follicles, which reside in the cortex of mammalian ovaries, remains one of the least understood processes of folliculogenesis, especially in humans and domestic animals. When cortical pieces from bovine and baboon ovaries are cultured in serum-free medium containing ITS+ (insulin-transferrin-selenium + bovine serum albumin and linoleic acid) wholesale, spontaneous activation occurs (i.e. primordial follicles initiate growth and develop into primary follicles). In contrast, we recently observed that activation does not occur when bovine cortical pieces are cultured in the absence of insulin (i.e. with TS+), but the follicles remain healthy; this provides a useful model for testing putative stimulators of follicle activation. The objective of this study was to test the hypothesis that kit ligand (Kitlg) stimulates activation of bovine primordial follicles in vitro. Small pieces of cortex were dissected from bovine fetal ovaries (6–8 months gestation; n = 3 fetuses) and cultured for 4 days (4 pieces/treatment/fetus) with 1) ITS+, 2) TS+ or 3) TS+ and Kitlg (10, 50, 100, 500 and 1000 ng/ml). After 4 days of culture, pieces were fixed, embedded in plastic and serially sectioned; every other set of 10 sections was mounted and stained with toluidine blue. One section in each mounted set was analyzed by histological morphometry to determine numbers and sizes of follicles. Only follicles in which the oocyte nucleus was visible were considered; this sampling method ensures that follicles are counted only once. Follicles in each section were classified as primordial, primary, or secondary and as healthy or atretic and the mean number of follicles per histological section was determined for each cortical piece. Pieces fixed on day 0 served as non-cultured controls. After 4 days of culture in TS+ the number (mean ± SEM) of primordial and primary follicles per histological section (8.8 ± 1.4 and 2.7 ± 0.4, respectively) did not differ from day 0 (8.7 ± 0.4 and 2.7 ± 0.6), indicating absence of activation in TS+. As expected, after 4 days cortical pieces cultured in ITS+ had very few primordial follicles (1.3 ± 0.4; P < 0.05 vs. day 0 and TS+) and the number of primary follicles per section had increased to 9.0 ± 1.0, approximately a 3-fold increase relative to day 0 and to TS+ (P < 0.05). When cortical pieces were cultured with TS+ and Kitlg, Kitlg at all doses tested decreased (P < 0.05) the number of primordial follicles and the four highest doses increased (P < 0.05) the number of primary follicles in a dose-dependent fashion, compared to day 0 or to TS+ alone, indicating that Kitlg stimulated activation of bovine primordial follicles. Interestingly, there were more primordial follicles and fewer primary follicles with all doses of Kitlg than with ITS+ (P < 0.05), showing that fewer follicles were activated by Kitlg than by the insulin in ITS+. There were no treatment effects on the mean number of follicles per section or on follicular health; the incidence of atresia in each follicle class was less than 20%. In conclusion, Kitlg stimulates activation of bovine primordial follicles in vitro in the absence of insulin. The fact that fewer follicles were activated by maximally effective doses of Kitlg compared to insulin suggests that the two factors stimulate activation via different mechanisms, but this hypothesis remains to be tested. (poster)

Germline Stem Cells and Follicular Renewal in the Postnatal Mammalian Ovary

Up to now, the accepted wisdom was that, unlike males who retain germline stem cells (GSCs) through their adult lives, oocyte production in females of most mammalian species ceases before birth. Females therefore are born with a fixed pool of GSCs, or oocytes, all of them arrested in meiosis. The ovarian follicles are oocytes enclosed by somatic cells. The oocyte reserve in human females is exhausted at approximately the fifth decade of life, inducing menopause. Directly contradicting these views, the investigators have found that juvenile and adult mouse ovaries have mitotically active germ cells that continuously replenish the pool of follicles. A study of mouse ovaries, based on analyses of healthy quiescent and early growing follicle numbers, showed that approximately one third of the peak endowment of immature follicles was lost by young adulthood. In contrast to previous studies in mammals, a parallel study of atretic (degenerating) follicles showed that atresia increased markedly by days 30 to 40, reaching a peak of more than 1200 dying follicles per ovary that continued well into reproductive life (Fig. 1). There was a clear discordance between changes in numbers of nonatretic follicles and the corresponding incidence of atresia in the postnatal mammalian ovary. Histologic studies demonstrated large ovoid cells that resembled germ cells of the fetal mouse ovary; they were found in the surface epithelial cell layer covering the ovary. Immunohistochemical studies affirmed that these cells were of a germline lineage. Immunostaining confirmed the presence of germ cells in varying stages of meiosis. The investigators identified genes whose expression is necessary for replicating germ cells to produce oocytes for follicle formation in postnatal life. In studies with busulphan, a germ-cell toxicant, exposed ovaries had fewer than 5% of the primordial follicle pool present in control animals. The rate of follicle loss was very close to that expected from previously reported data. Finally, ovarian fragments taken from wild-type mice were grafted into the ovaries of transgenic female siblings after removing approximately half of the host’s ovary. The transgenic germ cells infiltrated the grafted tissue and initiated folliculogenesis with the resident wild-type somatic cells. These observations suggest that GSCs are present in the postnatal mouse ovary. The findings have therapeutic potential for expanding the follicle reserve so as to postpone normal or premature ovarian failure.Fig. 1: Postnatal ovarian germ-cell dynamics. (A and B) Numbers of nonatretic (A) and atretic (B) primordial and total immature (primordial, primary, small preantral) follicles in mouse ovaries during postnatal development (B, total immature follicles; mean ± standard error, n = 3–4 mice per age group). (C) Incidence of primordial and primary follicle atresia in ovaries exposed to DMBA on day 25 postpartum (mean ± standard error, n = 4–5 mice per time point). (D) Comparison of nonatretic and atretic immature follicle numbers in C57BL/6, CD1, and AKR/J strains of mice (mean ± standard error, n = 3–5 mice per age group). Reproduced with permission. Nature 2004;428:145–150. Copyright © 2004, Nature Publishing Group. All rights reserved.

Ovarian follicle development requires Smad3.

Smad3 is an important mediator of the TGF beta signaling pathway. Interestingly, Smad3-deficient (Smad3-/-) mice have reduced fertility compared with wild-type (WT) mice. To better understand the molecular mechanisms underlying the reduced fertility in Smad3-/- animals, this work tested the hypothesis that Smad3 deficiency interferes with three critical aspects of folliculogenesis: growth, atresia, and differentiation. Growth was assessed by comparing the size of follicles, expression of proliferating cell nuclear antigen, and expression of cell cycle genes in Smad3-/- and WT mice. Atresia was assessed by comparing the incidence of atresia and expression of bcl-2 genes involved in cell death and cell survival in Smad3-/- and WT mice. Differentiation was assessed by comparing the expression of FSH receptor (FSHR), estrogen receptor (ER) alpha, ER beta, and inhibin alpha-, beta(A)-, and beta(B)-subunits in Smad3-/- and WT mice. Because growth, atresia, and differentiation are regulated by hormones, estradiol, FSH, and LH levels were compared in Smad3-/- and WT mice. Moreover, because alterations in folliculogenesis can affect the ability of mice to ovulate, the number of corpora lutea and ovulated eggs in response to gonadotropin treatments were compared in Smad3-/- and WT animals. The results indicate that Smad3 deficiency slows follicle growth, which is characterized by small follicle diameters, low levels of proliferating cell nuclear antigen, and low expression of cell cycle genes (cyclin-dependent kinase 4 and cyclin D2). Smad3 deficiency also causes atretic follicles, degenerated oocytes, and low expression of bcl-2. Furthermore, Smad3 deficiency affects follicular differentiation as evidenced by decreased expression of ER beta, increased expression of ER alpha, and decreased expression of inhibin alpha-subunits. Smad3 deficiency causes low estradiol and high FSH levels. Finally, Smad3-/- ovaries have no corpora lutea, and they do not ovulate after ovulatory induction with exogenous gonadotropins. Collectively, these data provide the first evidence that reduced fertility in Smad3-/- mice is due to impaired folliculogenesis, associated with altered expression of genes that control cell cycle progression, cell survival, and cell differentiation. The findings that Smad3-/- follicles have impaired growth, increased atresia, and altered differentiation in the presence of high FSH levels, normal expression of FSHR, and lower expression of cyclin D2, suggest a possible interaction between Smad3 and FSH signaling downstream of FSHR in the mouse ovary.

Association Of Single Umbilical Artery With Common And Rare Congenital Malformations

Single umbilical artery (SUA) is the most common abnormality of the umbilical cord. It is associated with an increased incidence of atresia of hollow organs, renal abnormalities, limb reduction defects and spontaneous abortions. The aetiopathogenesis, although unknown, is attributed to vascular disruption. In this study, 15 foetal autopsies with SUA, seen over a one-year period have been reviewed. Apart from the common associations, certain other abnormalities and syndromes, not described previously, have been identified. In addition to the well known multi-system malformations, five cases demonstrated unilateral or bilateral cystic renal dysplasia. Two of these cases had posterior urethral valves, whilst two cases showed additional features of Potter's sequence and one case showed Meckel's syndrome in association with SUA. These anomalies may have been due to obstruction to the urinary outflow, which is known to cause cystic renal dysplasia or may have resulted from ischaemia associated with SUA.

The yolkless egg and the evolution of eutherian viviparity.

All vertebrate follicles have the same basic structure. Viviparity also occurs in all vertebrates except birds, but it is the only form of reproduction in eutherians ("placental mammals"). Their mature follicles are vesicular, and their oocytes are yolkless. Clues to the origin of these unique characteristics are in the incidence of atresia and the role of yolk in reproduction. In broadcast spawning, atresia is as rare as it is common among eutherians and other nonspawning vertebrates. In all but the eutherians, at least the initial-and in most cases all-stages of embryogenesis depend crucially on the zygote's yolk. Eutherian reproduction, therefore, must have evolved in connection with genetic changes that caused fragility of the oocyte, instability of the follicle, and loss of the ability to produce vitellogenin (VTG), the main lipoprotein of yolk. Mutations can result in adaptations by uncovering hidden properties in a trait and/or its environment. Useful mutations in recessive alleles can spread through a population as heterozygotes, invisible until the number of homozygotes for the mutation is large enough for them to suddenly appear and form the nucleus of a new breeding population. Such a mutation probably truncated a long, oviductal-based, aplacental gestation of a small, lightly yolked zygote in an endothermic, mammal-like reptile and converted it into an early monotreme or marsupial-like mammal (pantothere). Against tremendous odds, another mutation later caused loss of the genes for VTG. The resultant yolkless zygote survived because 1). the mutation also affected a network of homeiotic genes controlling the ontogeny of the entire reproductive system and 2). the system contained enough hidden properties for the mutation to change the character of the oocyte, its granulosa cells and corpus luteum, the zygote, and the uterus in a way that virtually assured the new zygote's survival. Eutherian reproduction, however, is neither better nor worse than other forms; it is only different.

Immunization against 5α-androstane-3α,17β-diol affects ovarian folliculogenesis in Merino ewes

The 5α-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5α-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5α-androstane-3α,17β-diol (3α-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF 2α analog (PG, 125 mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24 h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2 mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17β (E 2), progesterone (P), androstenedione (A 4), testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α-ol,17β-one (androsterone: 3α-ol) and 3α-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5α-reduced substrates 3α-diol and 3α-ol but not to E 2, P, A 4 or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3α-ol and 3α-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5α-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5α-reduced substrates 3α-ol and 3α-diol at the ovarian level.

Quantification of the Ovarian follicles of water Buffaloes (&lt;i&gt;Bubalus bubalis&lt;/i&gt;)

Ovarian samples were collected either through ovariectomy or slaughtering of Philippine water buffaloes at the Philippine Carabao Research and Development Centre (now Philippine Carabao Centre), Central Luzon State University, Philippines. From the young buffalo (6 –7 months, 2 pairs of ovaries); pubertal (2 year; n=1 pair); adults (7 –8 year; n = 3 pairs) and old (12 – 14 years; n = 3 pairs) were used for the study. The ovaries were fixed, dehydrated and embedded in paraffin wax and were serially sectioned at 10 microns. The sectioned ovaries were then stained with periodic acid/schiffs-Hematoxylin (PAS-H). Ovarian follices of the following categories (primary, growing, secondary, tertiary and atretic) were quantified in the young, pubertal, adult and old water buffaloes. In the young and pubertal buffaloes, the average follicular population of 75,604 and 85,358 primary follicles were counted, respectively. The numbers of primary follicle decreased as age of the animal increased, meaning the follicles are either ovulated or become atretic during the course of the estrous cycle. The growing follicles were highest in the pubertal animals with an average follicle number of 7,317, which is 8.75 percent of the primordial follicles. The young and adult buffaloes had an average count of 71 and 18 growing follicles respectively. The tremendous increase in the number of growing follicles during the pubertal stage may indicate that follicle are continually being recruited and they grow from stage to stage and end life by becoming atretic or become ovulated. The number of secondary follicles found in each age group decreased with age. The high number of secondary follicles counted in the pubertal revealed that there was a transition of the growing follicles to the secondary stage. Further decline in the number of tertiary follicle occurred, while the average number for tertiary follicles in the young, adult and old were 3,9,7, respectively which is quite low. Data showed that there was a high incidence of atresia occurring in the ovarian follicles of buffaloes ranging from 127 in the young to 142 in the pubertal. Keywords: Quantification, Ovarian follicle and water buffaloes

Aryl hydrocarbon receptor regulates growth, but not atresia, of mouse preantral and antral follicles.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that binds various environmental contaminants. Despite our knowledge regarding the role of the AhR in mediating toxicity, little is known about the physiological role of the AhR. Previous studies indicate that the AhR may regulate folliculogenesis, because AhR-deficient (AhRKO) mice have fewer preantral and antral follicles than wild-type (WT) mice during postnatal life. Thus, the first objective of the present study was to test the hypothesis that AhR deficiency reduces the numbers of preantral and antral follicles by slowing growth and/or increasing atresia of follicles. Because alterations in follicular growth or atresia can affect the ability to ovulate, the second objective was to test whether AhR deficiency reduces the number of ovulated eggs. To test these hypotheses, follicular growth was compared in WT and AhRKO ovaries using morphometric techniques and by measuring the ability of the ovary and follicles to grow in response to eCG. Atresia was compared in WT and AhRKO ovaries using morphometric techniques, TUNEL assays, and 3'-end labeling of fragmented DNA. Ovulation was compared in WT and AhRKO mice by assessing the number of corpora lutea per ovary. The results indicate that follicular growth and ovulation were reduced in AhRKO ovaries compared to WT ovaries. The WT ovaries had a 1.5-fold increase in the number of preantral and antral follicles between Postnatal Days 32 and 45, were more responsive to eCG, and contained more corpora lutea than AhRKO ovaries. In contrast, no significant difference was observed in the incidence of atresia in WT and AhRKO ovaries. Taken together, these results suggest that the AhR may regulate growth, but not atresia, of preantral and antral follicles in the mouse ovary.

The influence of feeding regime on sexual maturation, fecundity and atresia in first‐time spawning turbot

Exposure of adolescent turbot Scophthalmus maximus to low rations during vitellogenesis, covering the 4 months immediately prior to spawning, led to a drop of 70% in mean ovary weight, and was associated with poor growth of the vitellogenic oocytes or, in a third of cases, the absence of vitellogenic oocytes. Exposure to low rations during the recruitment of vitellogenic oocytes, 4–8 months prior to spawning, produced a more variable response. A few of these females produced well‐developed vitellogenic oocytes, but in most, vitellogenic oocytes were absent or only poorly developed and in some cases there was a high incidence of atresia. Exposure to intermediate rations throughout oocyte recritment and vitellogenesis also depressed oocyte development. In maturing females the number of non‐atretic vitellogenic oocytes (relative potential fecundity) averaged 998 per g of somatic body weight of fish at the start of the spawning season in mid‐June. Maturation of the males was not significantly influenced by the dietary regime. The small size of the testes and the low levels of milt production, even in fish fed high rations immediately prior to spawning, showed that reproductive investment in males was much lower than in females.

Reproductive Condition and Behavior in Relation to Plasma Levels of Gonadal Steroids in the Spiny Damselfish Acanthochromis polyacanthus

Gonadal condition and plasma levels of gonadal steroids were measured in relation to behavior in the biparental brood-protecting spiny damselfish Acanthochromis polyacanthus. Fish were captured by SCUBA divers from natural populations on Australia's Great Barrier Reef and immediately bled underwater, following diver or video observation of precapture behaviour. In winter (July), most fish were nonterritorial, with a low proportion of mature males, or vitellogenic females present. In spring (November), most fish were territorial with broods of young at varying stages of maturity, and all stages of gonadal development were represented. Territorial males were larger than nonterritorial fish, but territorial fish that had large (older) young (the end of the brooding phase) had lower condition factors than fish at other stages. Males of all gonad stages had a high proportion of spermatozoa in the testis, but this was higher in November than in July. Ovaries of females commonly had several classes of developing follicles present, although fish that were brooding large young had regressed ovaries with a high incidence of atresia. Plasma levels of testosterone (T) and 11-ketotestosterone were elevated in males of advanced gonadal maturity, and also in relation to recent or imminent spawning behaviour, but there were no changes in plasma 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), which was near assay detection limits at all times. Females had elevated T and 17β-estradiol (E2) in association with vitellogenesis and elevated T in relation to spawning activity in some fish, but as in males, 17,20βP levels were low and unchanging. Territorial females without young had lower cortisol levels than nonterritorial fish, or females protecting young. The results confirm the importance of elevated androgens to spawning activity in territorial male fish, but not females where endocrine activity is more closely related to stage of ovarian development. Extended brooding appears to inhibit vitellogenesis, perhaps via a stress-related mechanism.

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17β-estradiol, 17α-20β-dihydroxy-4-pregnen-3-one (17α-20β-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17β-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17β-estradiol levels. Inversely, plasma 17α-20β-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17α-20β-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17β-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.

Effect of passive immunization with buffalo follicular fluid antisera on ovarian activity in guinea pigs.

Ovarian activity and follicular populations were studied in guinea pigs (Cavia porcellus) following administration of antisera against buffalo follicular fluid (buFF). Antibodies were raised in rabbits and the titre tested by immunodiffusion assay. Fourteen guinea pigs cycling normally were randomized into two groups. Animals in Group I (n = 8) were treated (i.p.) with 0.5 ml antisera and in Group II (control, n = 6) with the same volume of normal rabbit serum at 12 h intervals on the 10th and 11th day of their oestrous cycle. They were sacrificed 24 h after onset of estrus when ovulation points were counted and ovaries processed for microscopical examination. Treatment with buFF-antisera increased ovulation rate (3.6 vs. 2.0; p < 0.01) but had no significant effect on the total number of follicles. However, the treatment reduced the percentages of atretic follicles in all size classes. These results indicated that the administration of a buFF-antisera produced in the rabbits increased ovulation rate in guinea pigs by reducing the incidence of atresia.

Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat (Capra hircus).

The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat's estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period (day 0 = day of sponges withdrawal), 2 of the goats were bilaterally ovariectomized. Follicles with a diameter > 1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5 +/- 9.8 h. LH peak concentration averaged 44 +/- 5.3 ng/ml and the mean length of the preovulatory surge (amounts over 10 ng/ml) was 8.9 +/- 0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles (58 vs. 30, P < 0.05). The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles (23 vs. 6, P < 0.05). On day 2, the number of advanced atretic follicles was greater than early atretic follicles (10 vs. 4, P < 0.05). There was an increase in the number of early atretic follicles from day 2 to day 4 (4 vs. 9, P < 0.05), which was consistent with the effects of the preovulatory LH surge.

Steroidogenic enzyme expression during preovulatory follicle maturation in pigs.

During preovulatory maturation, follicles destined to ovulate exhibit a marked increase in steroidogenesis, while nonovulatory, steroidogenically inactive follicles are lost by atresia. The purpose of this study was to assess the expression of the steroidogenic enzymes cytochrome P450 aromatase (P450arom), cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (P450c17), and 3beta hydroxysteroid dehydrogenase (3betaHSD) and that of the cell proliferation-associated nuclear antigen Ki-67 by immunohistochemistry in both nonatretic and atretic follicles developed during altrenogest-synchronized preovulatory maturation. Fifteen cyclic gilts were slaughtered on Days 1, 3, 5 (n = 3 per day), and 7 (n = 6) following the withdrawal of altrenogest. Staining intensity was assigned a numeric value and was related to follicle size and day following altrenogest withdrawal. Follicle atresia was assessed by the extent of in situ 3' end-labeling of DNA in apoptotic cells. The overall incidence of atresia was 41%, 17%, and 7% in small (< 3 mm), medium (3-5 mm), and large (> 5 mm) follicles, respectively. Expression of granulosa cell P450arom, Ki-67 antigen, and thecal P450c17 was significantly less (p < 0.001) in atretic compared to nonatretic follicles. Thecal 3betaHSD was expressed predominantly in medium and large nonatretic follicles; expression was nondetectable in 88% of small follicles irrespective of follicle health. Ki-67 expression in granulosa cells was greater in small and medium nonatretic than in large nonatretic follicles (p < 0.005). In large presumptive ovulatory follicles, P450arom was maximally expressed on Days 3 and 5 and decreased on Day 7 (p < 0.005), while P450c17 was unchanged between Days 3 and 7. In contrast, 3betaHSD increased linearly between Days 3 and 7 (p < 0.005). Staining intensities of P450arom, P450c17, and 3betaHSD were correlated with each other and with follicular fluid concentrations of the steroids estradiol, androstenedione, and progesterone, whose production they catalyzed. These data show that selection of ovulatory follicles is associated with a low incidence of apoptosis, a reduction in cell proliferation, maintenance of high levels of P450arom, and increased expression of 3betaHSD to provide substrate for androgen and estrogen production. During the period of development investigated in this study, changes in follicular steroid production in vivo are explained in large measure by changes in steroidogenic enzyme expression.

Expression of androgen receptors and steroidogenic enzymes in relation to follicular growth and atresia following ovulation in pigs.

Among the new antral follicles that develop after ovulation in pigs, the incidence of atresia, based on granulosa cell apoptosis, increases between Days 5 and 7 of the estrous cycle. The purpose of this study was to determine how follicular growth and atresia affected the expression of some key enzymes regulating follicular steroidogenesis and androgen receptor on Days 3, 5, and 7 after the onset of estrus. Ovaries were frozen in liquid propane for subsequent sectioning and immunohistochemical analysis. Ninety-six follicles were classified according to size as small (< 3 mm), medium (3-5 mm), or large (> 5 mm). Follicles in the active stages of the cell cycle were identified by the presence of the cell proliferation-associated nuclear antigen Ki-67 in granulosa cells. Follicles with apoptotic cells were identified by in situ 3'-end labeling of DNA. Staining intensity of antigens on sections was assigned a numeric value (0-3). Follicles assigned a value > 1 for 3'-end labeling in their granulosa cells were classified atretic. The percentage of atretic follicles increased (p < or = 0.05) from 5% on Days 3 and 5 to 41% on Day 7. Expression of Ki-67 in granulosa cells was more strongly (p < or = 0.05) associated with nonatretic follicles (98% expressing) than with atretic follicles (41% expressing). Aromatase cytochrome P450 (P450arom) was localized predominantly in granulosa cells of nonatretic follicles and was undetectable in atretic follicles. Androgen receptor in granulosa cells and expression of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) in theca interna were lower (p < or = 0.001) in atretic follicles than in nonatretic follicles. The expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was localized to the theca interna and was unaffected by follicle atresia. In nonatretic small follicles, the expression of P450arom and P450c17 decreased (p < 0.01) between Days 3 and 7 while expression of Ki-67 was unchanged. In nonatretic follicles, increased follicle size was associated with a decrease (p < 0.01) in androgen receptor expression and increases (p < 0.01) in P450arom, P450c17, and 3 beta HSD expression. In conclusion, increased expression of steroidogenic enzymes was associated with follicular growth. Loss of P450arom expression in vivo is an early event in atresia and is followed by decreased cell proliferation, and decreased expression of androgen receptor and P450c17.

Follicular Atresia, Follicular Fluid Hormones, and Circulating Hormones During the Midluteal Phase of the Estrous Cycle in Pigs1

The incidence of atresia, concentrations of follicular fluid steroids, and circulating concentrations of estradiol, FSH, and progesterone were investigated to determine whether there was any evidence for a wave of follicular activity or atresia between Days 7 and 15 of the estrous cycle in pigs, Cyclic pigs (gilts) were slaughtered on Days 7, 9, 11, 13, and 15 (4, 4, 4, 4, and 3 pigs per day), and 287 follicles > 2 mm were dissected from ovaries to recover follicular fluid and granulosa cells. Apoptotic (Ao) granulosa cells were those containing sub-diploid DNA fluorescence as determined by flow cytometry. follicles with > or = 10% Ao granulosa cells were classified as atretic, and those with < 10% Ao granulosa cells were designated nonatretic. The percentage of atretic follicles (AF) per pig was 35.5 +/- 4.7 (mean +/- SEM), and while the percentage of AF ranged from 12% to 73%, means did not differ significantly among days. Mean follicular estradiol concentration was higher (p < 0.05) in nonatretic follicles (NAF) than in AF; in NAF, the concentration decreased (p < 0.05) from 15 ng/ml on Day 7 to 2.5 ng/ml on Day 11 and then increased (p < 0.05) to 10 ng/ml on Day 15 of the cycle. Mean follicular progesterone concentration was higher (p < 0.05) in AF than in NAF, while follicular androstenedione concentration (20 +/- 1.5 ng/ml, overall mean +/- SEM) did not differ between AF and NAF. Mean plasma estradiol, FSH, and progesterone concentrations did not differ among days of the cycle or differ significantly during the last 42 h before slaughter. Plasma estradiol and FSH concentrations were not significantly correlated with percentage of AF or with follicular steroid concentrations. These results support the notion of continuous growth and atresia of ovarian follicles in pigs independent of changes in plasma FSH and follicular steroid concentration, without evidence for a dominant cohort of follicles during the luteal phase of the estrous cycle.

Effects of Gonadotropin Treatment and Withdrawal on Follicular Growth, Cell Proliferation, and Atresia in Ewes1

To determine the effects of FSH-P treatment and subsequent withdrawal on follicular growth, cell proliferation, and atresia, ewes (n = 4 ewes/treatment group) received twice daily injections of saline or FSH-P beginning on Day 13 of the estrous cycle (length of the estrous cycle = 16.5 days) and were slaughtered after 0, 48, or 72 h of treatment (i.e., on Days 13, 15, or 16). Some treatment groups received FSH-P from Day 13 until slaughter (FSH-P-treated), whereas some received FSH-P for 24-48 h followed by saline for 24-48 h (FSH-P withdrawal). All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analogue) 1 h before slaughter. For both ovaries from each ewe, the number and surface diameter of all visible antral follicles were determined, and antral follicles were classified as small (< or = 3 mm), medium (> 3 mm to < or = 6 mm), or large (> 6 mm). As an index of the rate of cell proliferation, BrdU was immunolocalized in paraffin-embedded tissue sections, and the labeling index (LI; BrdU-labeled nuclei as a percentage of total nuclei) was determined for granulosa and thecal cells of nonatretic and early atretic antral follicles of known diameter. Follicular status (atretic vs. nonatretic) was evaluated morphologically. Moreover, the presence of apoptosis was detected in situ by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method. For untreated and saline-treated ewes, the number of small follicles per ewe increased (p < 0.01) from Day 13 to Day 15, then decreased again on Day 16, whereas numbers of medium and large follicles did not differ across days. Compared with saline-treated ewes, ewes receiving FSH-P from Day 13 until slaughter had fewer (p < 0.05) small but more (p < 0.05) medium and large follicles. Compared with FSH-P-treated ewes, FSH-P withdrawal resulted in fewer (p < 0.05) medium and large but more (p < 0.05) small follicles. Across all follicular size classes, granulosa and thecal cell LI of nonatretic follicles was decreased (p < 0.05) by FSH-P withdrawal compared with FSH-P treatment. Additionally, across all follicular size classes, FSH-P withdrawal increased (p < 0.01) the percentage of follicles that were atretic compared with saline or FSH-P treatment. Histochemical staining of early and advanced atretic follicles showed that granulosa cells are the predominant site of cell death (apoptosis) during follicular atresia. Thus, compared with continuous FSH-P treatment, withdrawal of FSH-P resulted in decreased numbers of medium and large follicles, decreased proliferation of follicular cells, and an increased incidence of atresia associated with granulosa cell death. This model should prove useful for studying the mechanisms regulating follicular growth and atresia in ewes.

Changes in insulin-like growth factor-binding protein-2 and -3 in follicular fluid during atresia of follicles grown after ovulation in pigs.

In pigs, medium-sized follicles (3-6 mm in diameter), lost during preovulatory maturation, are rapidly replenished after ovulation, increasing tenfold in number between day 3 and day 8 of the oestrous cycle. The incidence of atresia in medium-sized follicles was previously found to increase between day 5 and day 7 after the onset of oestrus (day 0). This study was conducted to determine whether changes in concentrations of insulin-like growth factor binding proteins (IGFBPs) in pig follicular fluid from medium-sized follicles were related to increased incidence of atresia. Individual follicles were isolated on days 5, 6 and 7 after oestrus from eleven pigs. Follicles were defined as atretic when they contained > or = 10% apoptotic granulosa cells (cells containing subdiploid amounts of DNA fluorescence determined by flow cytometry). Concentrations of IGFBP-2 and -3 were determined by ligand blot analysis with 125I-labelled insulin-like growth factor II and quantified by densitometry. The mean percentage of apoptotic cells per follicle increased (P < or = 0.01) from 3.6 to 35.0 and the mean concentration of IGFBP-2 increased (P < or = 0.001) 3.3-fold between day 5 and day 7 after oestrus. The concentration of IGFBP-3 did not differ significantly among days. Mean oestradiol concentration in follicular fluid decreased (P < or = 0.05) from 52.3 to 0.9 ng ml-1 between day 5 and day 7. Androstenedione concentrations in follicular fluid also decreased (P < or = 0.01) between day 5 and day 7, but progesterone did not differ significantly among days.(ABSTRACT TRUNCATED AT 250 WORDS)