Stimulatory and inhibitory effects of progesterone on follicular development in the hypophysectomized follicle-stimulating hormone/luteinizing hormone-treated hamster

Cyclic hamsters hypophysectomized at estrus (Day 1 of the cycle) and injected with 5 micrograms follicle-stimulating hormone (FSH) on Day 1 and 20 micrograms luteinizing hormone (LH) in polyvinylpyrrolidone (PVP) from Days 1-4 ovulated 15.3 ova, in response to 30 IU human chorionic gonadotropin (hCG) administered at 1500 h on Day 4 (Kim and Greenwald, 1984). When 1 mg progesterone (P4) was administered daily from Days 1-4 concurrent with the above regimen, ovulation increased to 38 ova, a clearcut superovulatory response. However, daily injection of 1, 10, or 100 micrograms P4 plus FSH and LH reduced the number of antral follicles present on the afternoon of Day 4 to 3-4 per ovary, compared to 9 per ovary after FSH-LH alone, and the ovulation rate was drastically reduced with most animals being anovulatory. Substituting 1 mg 17 alpha-hydroxyprogesterone or estradiol cyclopentylpropionate for P4 on Days 1-4 did not alter the number of antral follicles on Day 4 from FSH-LH alone, whereas 1 mg androstenedione or 1 mg testosterone cyclopentylpropionate reduced the number of antral follicles to 3 or less. Hence, the stimulatory effects of 1 mg P4 are not attributable to its conversion to other P4 derivatives. After the concurrent injection of 1 mg P4 and FSH-LH, on the afternoon of Day 3, an average of only 1.8 large preantral follicles was present per ovary. By the morning of Day 4, however, the ovary contained 14 large preantral and early antral follicles in addition to 8 large antral follicles. Injection of hCG at this time resulted in the ovulation of 14.5 ova.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-type-specific localization of transforming growth factor-beta 2 and transforming growth factor-beta 1 in the hamster ovary: differential regulation by follicle-stimulating hormone and luteinizing hormone.

Cell-type-specific localization and gonadotropin regulation of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) in the hamster ovary were evaluated immunohistochemically under three conditions: (1) during the estrous cycle (Day 1 = estrus; Day 4 = proestrus); (2) after the blockade of periovulatory gonadotropin surges by phenobarbital, and (3) after FSH and/or LH treatment of long-term hypophysectomized hamsters. Ovarian TGF-beta 1 activity was primarily localized in theca and interstitial cells. The activity increased moderately but significantly after the preovulatory LH surge and reached a peak at 0900 h, Day 2 h; oocytes showed considerable activity. TGF-beta 1 immunoreactivity subsequently fell to low levels in theca-interstitial cells through 0900 h, Day 4. Significant TGF-beta 2 immunoreactivity appeared after the surge, mainly in the granulosa cells of both preantral and antral follicles; a few interstitial cells surrounding preantral follicles showed discrete staining. TGF-beta 2 immunoreactivity in granulosa cells and in interstitial cells next to preantral follicles reached a peak at 0900 h, Day 1, and persisted up to 0900 h, Day 2; oocytes showed no staining. Phenobarbital treatment blocked the appearance of TGF-beta 1 and TGF-beta 2 immunoreactivities at 1600 h, Day 4; however, a rebound in immunoreactivities was observed with the onset of the surge after a 1-day delay. Replacement of LH to long-term hypophysectomized hamsters resulted in a marked increase in TGF-beta 1 immunoreactivity in the interstitial cells, but FSH, although it induced follicular development, did not influence ovarian TGF-beta 1 activity. Treatment with FSH, however, induced a massive increase in TGF-beta 2 immunoreactivity in the granulosa cells of newly developed antral and preantral follicles but not in the interstitial cells; LH, on the other hand, had no significant effect on TGF-beta 2 activity. Treatment with FSH and LH combined resulted in a dramatic increase in TGF-beta 2 immunoreactivity in granulosa and interstitial cells and in TGF-beta 1 in theca and interstitial cells comparable to their peak activity in intact animals. Western analyses substantiated the presence of TGF-beta 1 and TGF-beta 2 in the hamster ovary and the specificity of immunolocalization. These studies, therefore, provide critical evidence that TGF-beta 1 and TGF-beta 2 in the hamster ovary are expressed in specific cell types and that their expression is differentially regulated by LH and FSH, respectively.     

Hormonal changes associated with estrogen induced luteolysis in the pregnant hamster: a reevaluation of the luteotropic complex

Hamsters were injected sc on Day 1 of pregnancy (sperm positive) with 50 micrograms estradiol cyclopentylpropionate (ECP) or peanut oil. On Day 5, serum progesterone (P4) was 10.6 ng/ml in controls vs 3.1 ng/ml after ECP. In the ECP group, serum prolactin (PRL) and follicle stimulating hormone (FSH) did not differ from controls but serum luteinizing hormone (LH) was significantly lower than that of the controls, and usually below the sensitivity of the radioimmunoassay (RIA). After ECP, structural signs of luteolysis (weight and histology) and absence of antral follicles characterized the ovary. Injection of an anti-LH serum on Day 4 halved serum P4 levels on Day 5 in control animals but caused no further lowering of P4 in ECP-treated hamsters. Treatment on Days 1-5 with 1.0 IU hCG or 10 micrograms LH plus ECP on Day 1 restored, by the afternoon of Day 5, serum P4 to the control range (9-10 ng/ml) and antral follicles were now present. The results indicate that a large dose of ECP causes luteolysis by reducing LH levels and reinforce the concept of a luteotropic complex in the hamster with PRL and FSH constituting the minimal components and LH serving as a synergist.     

In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin.

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media-before and after gonadotropin administration-were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 microm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-microm and 400-microm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 microm in diameter with 4-5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110-148 microm in diameter, had 2-4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1-4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39-46). The technique developed to collect intact rat follicles should be useful for numerous investigations.     

A species difference between hamster and rat in the effect of oestrogens on growth of large preantral follicles

Intact or hypophysectomized 23-day-old hamsters and rats were injected s.c. with 2 mg diethylstilboestrol (DES) or 1 mg oestradiol cyclopentylpropionate (OECP) on Days 23-25 and killed on Day 26. Although serum oestradiol was elevated to the same high levels by OECP, ovarian and uterine weights were increased in the rat by OECP or DES whereas only the uterus responded in the hamster. This correlated with the ability of the oestrogens to increase significantly the number of large preantral and antral follicles in the intact rat but only the number of follicles with 2-3 layers of granulosa cells in the immature hamster. Qualitative study revealed that DES and OECP increased the number of large preantral follicles in the adult hypophysectomized rat but were ineffective in the adult hamster. It is concluded that for the immature and adult hamster oestrogens do not play a major role in the recruitment of large preantral follicles.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of melatonin and hypothyroidism on estradiol and gonadotropin levels in female Syrian hamsters

Since melatonin injections administered near the end of the daily photoperiod influence both gonadal and thyroid hormones in the female hamster, the present study was designed to compare the effects of melatonin and hypothyroidism on the reproductive system and to determine whether thyroid status influenced the action of melatonin on the regulation of the hormones of reproduction. The effects of daily melatonin injections were determined in control hamsters, in hamsters rendered hypothyroid with thiourea, and in hypothyroid hamsters receiving thyroxin (T4) hormone replacement. As previously reported, melatonin injections disrupted estrous cyclicity, disrupted the normal pattern of gonadotropin secretion, and resulted in atrophy of the uterus and vagina. These changes coincided with depressed serum and pituitary prolactin (PRL), and depressed levels of estradiol. The effects of melatonin on uterus, vagina, ovary, and on gonadotropin levels were not prevented by T4 replacement, with the exception of a melatonin-induced increase in serum follicle-stimulating hormone (FSH). This suggested that the cessation of estrous cyclicity was not primarily a result of thyroid deficiency. Hypothyroidism, however, like melatonin, resulted in a reduced number of developing and mature follicles and corpora lutea in the ovaries, and in reduced uterine weight. It also produced follicular atresia, reduced the circulating levels of estradiol, and resulted in reduced incidence of estrus smears. T4 replacement, for 2 weeks, prevented the decline in mature follicles and corpora lutea, reduced the extent of follicular atresia, increased circulating levels of estradiol, and increased uterine weight. PRL and luteinizing hormone (LH) data also provided evidence for antagonistic effects of melatonin and T4 in female hamsters. These data raise the question whether melatonin-induced changes in circulating levels of T4 play a role in the seasonal cycles of reproductive competence in the female hamster.     

Relationship between the appearance of preantral follicles in the fetal ovary of Antarctic minke whales (Balaenoptera bonaerensis) and hormone concentrations in the fetal heart, umbilical cord and maternal blood

The present study aimed to determine the relationship among changes in the number of preantral follicles and concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4), androstenedione (A) and estradiol-17beta (E2) in the fetal heart, umbilical cord and maternal blood. Primordial follicles had already appeared in a 20 cm fetus and primary follicles were observed in a 50 cm fetus. In a 70 cm fetus, the number of primordial and primary follicles increased rapidly and secondary follicles were present. The concentrations of LH and FSH did not change between 20 cm and 160 cm in fetal length. When the fetal length became > 70 cm, serum levels in the fetus, umbilical cord and mothers, and E2 levels in umbilical cord increased synchronously (p < 0.05). These results showed increases in the number of preantral follicles in the Antarctic minke whale fetal ovary along with fetal growth during the early gestation period. These findings suggest that the change in preantral follicles was associated with changes in the concentration of steroids in early gestation periods. The changes in steroid concentrations in the fetal and umbilical cord blood and the increased number of preantral follicles were coincident at around 70 cm in fetal length, whereas the growth and differentiation of primordial and primary follicles appeared to be independent of FSH and LH.     

Unilateral ovariectomy restores ovulatory cyclicity in rats with a polycystic ovarian condition

An acyclic polycystic ovarian condition can be induced in adult rats with a single injection of estradiol valerate (EV). The ovaries are small and contain multiple cystic follicles and no new corpora lutea. In the early stages of the condition, both basal plasma luteinizing hormone (LH) and LH responses to luteinizing hormone-releasing hormone (LHRH) are attenuated. Plasma androgens are indistinguishable from normal controls. The present study examines the effect of unilateral ovariectomy (ULO) on this condition. Removal of one cystic ovary results in almost immediate resumption of vaginal cyclicity that persists for at least 3 wk. At 1 or 3 wk after ULO the remaining ovary contains fresh corpora lutea, appears histologically normal, and is significantly heavier than the cystic ovary removed at ULO, indicative of compensatory hypertrophy. Despite the resumption of apparently normal cyclic function, basal plasma LH concentrations and LH responses to LHRH are not significantly better than those in intact animals with polycystic ovaries. Thus, the previously polycystic ovary is fully capable of normal ovulatory function despite obvious impairments in the hypothalamo-pituitary axis. Since ovulatory function resumes on a background of continued poor pituitary responsiveness, the primary defect, which ULO corrects, is probably at the hypothalamic level. Finally, the cystic ovary clearly contributes to the hypothalamic aberration to which it subsequently responds.     

An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects

An enzymatic method was developed to collect intact follicles at different stages of development from cyclic hamsters to study ovarian folliculogenesis under various circumstances. Ovaries from 6 adult hamsters on each day of the cycle (Day 1 = ovulation) were collected, corpora lutea and large preantral and antral follicles were dissected, and follicles saved. Minced ovaries were then incubated with a mixture of collagenase, DNAse and pronase at 37 degrees C for 20 min to disperse intact follicles. Histological studies with 2191 isolated follicles revealed 10 different stages of follicular development (depending on the number of granulosa cell layers surrounding the oocyte and development of the antrum). Of the total follicular population, 14% showed signs of atresia, with 50% of those having 1-3 layers of granulosa cells (Stages 1-3); a second peak of 18% was observed in antral follicles (Stages 8-10). No signs of thecal cells were evident until the follicles reached Stage 6 (7-8 layers of granulosa cells), which possibly accounts for reduced atresia in this class and beyond. Ultrastructural study revealed that there were no signs of morphological damage to the basement membrane or to other subcellular organelles in the small preantral follicles. The presence of subnuclear lipid droplets in follicles with 3 layers of granulosa cells provided evidence for potential steroidogenesis by small follicles. The number of Stage 1-10 follicles was remarkably constant throughout the estrous cycle (460 +/- 34 per animal on Day 1 vs. 492 +/- 66 on Day 4). The usefulness of this method in analyzing follicular kinetics is illustrated in experiments involving hypophysectomy and the effects of unilateral ovariectomy. This procedure offers an improved method to study the factors responsible for the growth and the differentiation of small preantral follicles in the mammalian ovary.     

Hormonal regulation of ovarian cellular proliferation

The steroid hormone estradiol, and the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are known to be essential for the growth and differentiation of follicles in the ovary. The present study was conducted to determine quantitatively the effects of estradiol, FSH and LH on proliferation of different ovarian cell types (granulosa and theca cells). The immature female hypophysectomized rate sequentially primed with estradiol, FSH and LH was used as the experimental model. Proliferation was assessed by examining changes in total DNA, incorporation of 3H-thymidine into DNA and labeling index in specific cell types. Estradiol and FSH each acted on follicles at different stages of development to stimulate proliferative activity of both granulosa and theca cells. Continued administration of either hormone caused a decrease in the proliferative activity of both cell types. These observations have been interpreted to indicate that estradiol and FSH can each alter the length of the specific phases of the cell cycle. A luteinizing dose of LH caused a cessation of proliferation in luteinizing granulosa cells while stimulating a limited proliferation of theca cells. Absence of the appropriate hormonal stimulus caused both granulosa and theca cells to stop proliferating and the follicles to undergo atresia. These results indicate that, depending upon the state of differentiation of granulosa and theca cells, estradiol, FSH and LH can stimulate or inhibit the ability of these cells to proliferate.     

Examination of the role of follicle stimulating hormone in estrogen biosynthesis in vivo and in vitro in the ovary of the cyclic hamster

The effect of neutralizing endogenous follicle stimulating hormone (FSH) or luteinizing hormone (LH) with specific antisera on the in vivo and in vitro synthesis of estrogen in the ovary of cycling hamster was studied. Neutralization of FSH or LH on proestrus resulted in a reduction in the estradiol concentration of the ovary on diestrus-2 and next proestrus, suggesting an impairment in follicular development. Injection of FSH antiserum at 0900 h of diestrus-2 significantly reduced the ovarian estradiol concentration within 6--7 h. Further, these ovaries on incubation with testosterone (T) in vitro at 1600 h of the same day or the next day synthesized significantly lower amounts of estradiol, compared to corresponding control ovaries. Although testosterone itself, in the absence of endogenous FSH, could stimulate estrogen synthesis to some extent, FSH had to be supplemented with T to restore estrogen synthesis to the level seen in control ovaries incubated with T. Lack of FSH thus appeared to affect the aromatization step in the estrogen biosynthetic pathway in the ovary of hamster on diestrus-2. In contrast to this, FSH antiserum given on the morning of proestrus had no effect on the in vivo and in vitro synthesis of estrogen, when examined 6--7 h later. The results suggest that there could be a difference in the need for FSH at different times of the cycle. Neutralization of LH either on diestrus-2 or proestrus resulted in a drastic reduction in estradiol concentration of the ovary. This block was at the level of androgen synthesis, since supplementing testerone alone in vitro could stimulate estrogen synthesis to a more or less similar extent as in the ovaries of control hamsters.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran reduces growth and disrupts reproductive parameters in female rats

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) are widespread environmental pollutants. TCDD is well known for its adverse effects on female reproduction when administered acutely to immature or adult rats. It is also known that fetal/neonatal exposure to this compound alters reproductive parameters. It is unknown whether exposure to PCDF causes similar adverse effects in offspring. The objectives of the study were to investigate the effects of in utero and lactational (IUL) exposure to TCDD and PCDF on subsequent growth, estrous cycles, and ovulation. Additionally a gonadotropin-primed immature rat model was used to investigate possible direct effects on the ovary after IUL exposure to TCDD (2.5 microg/kg) by evaluating 1) ovarian morphometrics and 2) serum estradiol concentrations. Body weights were reduced in animals with IUL exposure to TCDD and PCDF relative to those in controls at 10 days of age (P < 0.05 for each), and this difference was maintained until termination of the experiment at 125-165 days of age (P < 0.05). Exposure to TCDD or PCDF also disrupted regular estrous cycles and inhibited ovulation rate. On Day 23 (before eCG stimulation), ovaries from animals exposed to TCDD contained the same number of primordial, primary, secondary, preantral, and antral follicles as ovaries from control animals. On Day 25 (48 h after eCG stimulation), ovaries from TCDD-exposed rats had significantly fewer large preovulatory follicles when compared with ovaries from controls. The numbers of smaller follicles (both antral and small antral) were not different. Serum estradiol was significantly lower in TCDD-exposed animals 48 h after eCG stimulation.     

Hormonal regulation of activities of 4-ene-5 beta and 5 alpha-reductases and 17 beta-ol-dehydrogenase in immature golden hamster ovary

Diethylstilbestrol (DES) pellets were implanted in female golden hamsters on day 22 after birth. Hamsters with or without the DES pellet were hypophysectomized on day 23. Starting from day 26, the hypophysectomized hamsters were injected daily with 2.3-40 micrograms NIH-LH-S19, 6 or 18 micrograms NIAMD-oFSH-13, 50 micrograms NIAMD-Rat-FSH-B-1, or saline for 3 days. Ovarian homogenates from these hamsters on day 29 were incubated with [14C]-4-androstene-3,17-dione and enzyme activity (nmol/g/h) was estimated. The 5 alpha- and 5 beta-reductase activities decreased significantly following hypophysectomy. In the hypophysectomized hamster ovary, a distinct response to LH but not to FSH or DES in the 5 alpha-reductase activity was found. On the other hand, the 17 beta-ol-dehydrogenase activity was stimulated by FSH but not by LH or DES. The 5 beta-reductase activity was stimulated by DES, FSH or 2.3 micrograms LH but not by 7-40 micrograms LH. In the DES-treated, hypophysectomized hamster ovary, LH and FSH stimulated the 5 alpha-reductase and 17 beta-ol-dehydrogenase activities, respectively, but FSH or LH treatment had no significant effect on the 5 beta-reductase activity. These results show that the 5 alpha-reductase activity is regulated by LH, while the 17 beta-ol-dehydrogenase activity is stimulated by FSH in immature golden hamster ovary. The 5 beta-reductase activity seems to be regulated predominantly by FSH but the effect of FSH is largely mediated by estrogen.     

Follicular growth and intraovarian and extraovarian oocyte release after daily injections of melatonin and 6-chloro-melatonin in the Syrian hamster

Groups of adult female Syrian hamsters (Mesocricetus auratus) were injected daily at 17:00 hr with 2.5, 15, or 25 micrograms of melatonin (Mel) or 6-chloro-melatonin (Cl-Mel) for 12 weeks. An ovary from each animal was completely serially sectioned for light microscopic investigation. Judging from the presence of corpora lutea, there were some animals in each group that continued to cycle, although the postestrous, white mucous discharge had disappeared. Noncycling animals were most often found in the 25-micrograms group of Cl-Mel. Only uterine weights of noncycling animals treated with either 25 or 15 micrograms of Mel or Cl-Mel were statistically significantly depressed versus controls. Cl-Mel (25 micrograms) significantly suppressed the total number and size of antral follicles (P less than 0.05). Follicular ruptures with incomplete or complete release of the oocyte out of the follicular compartment were observed. The oocyte release occurred either into the ovary ("intraovarian oocyte release: IOR") or outside of the ovary ("extraovarian oocyte release: EOR"). Compared with controls, the total number of IOR was increased in all experimental groups with the exception of the 2.5-micrograms group of Cl-Mel. IOR appeared in both preantral and antral follicles, and often IOR was complete. In controls, only preantral follicles were involved in IOR; these were primarily incomplete ones. IOR was seen in cycling and noncycling animals. By contrast, EOR was exclusively observed in noncycling hamsters. It is concluded that the cessation of postestrous, white mucous discharge is not necessarily an index for a halt in cyclic ovarian function. Injections of 25 micrograms of Cl-Mel are more effective than 25 micrograms of Mel in suppressing ovarian function. Both Mel and Cl-Mel increase the frequency of IOR. Finally, noncycling hamsters show EOR that is regarded as an abnormal ovulation.     

Recovery of large preantral follicles from buffalo ovary: effect of season and corpus luteum

Preantral follicle can be considered as an alternative source of oocyte for in vitro production of embryos. The objective of the present study was to standardize a procedure for the isolation of large preantral follicles (>150-500 microm) from buffalo ovaries and to determine the effect of season and the presence of corpus luteum on the recovery rate of the large preantral follicles. A combined enzymatic cum mechanical approach was adopted to recover the large preantral follicles. In the first experiment, the ovarian cortical pieces were suspended in trypsin (1000-1500 BAEE units for milligrams of solid) and incubated at various temperatures for different periods, i.e. (1) trypsin (1%), 37 degrees C for 10 min; (2) trypsin (1%), 37 degrees C for 10 min + 4 degrees C for 3 h; (3) trypsin (0.5%), 37 degrees C for 20 min; (4) trypsin (0.25%), 37 degrees C for 20 min. Although there was no significant difference (P>0.05) among the different protocols, the first protocol yielded more follicles (3.2, 2.6, 1.8 and 1.5 per ovary, respectively). Hence, the first protocol was selected and used in the second and third experiments. In the second experiment, the effect of season, i.e. peak breeding season (October-March) versus low breeding season (April-September) was evaluated on the recovery rate of the large preantral follicles. The recovery rate of large preantral follicles from the ovaries during the peak breeding season was significantly (P<0.05) greater (9.92+/-0.85 per ovary) than that of the low breeding season (4.95+/-0.27 per ovary). In the third experiment, effect of the presence of corpus luteum on the recovery rate of large preantral follicles was studied. There was a significantly (P<0.05) higher yield of large preantral follicles from the ovaries with corpus luteum (8.05+/-0.88 per ovary) than for the ovaries without corpus luteum (4.57+/-0.43 per ovary). This study confirms that the large preantral follicles can be isolated from buffalo ovaries using a combination of enzymatic cum mechanical methods and that more large preantral follicles can be recovered during the peak breeding season and from the ovaries having corpus luteum.     

Genetic differences in follicular DNA synthesis between two strains of hamsters

This study was designed to compare our previous results on ovarian follicular DNA synthesis by hamsters obtained from Sasco Laboratories with a different breeding colony: Harlan. Follicles from proestrous Harlan hamsters required twice as much [3H] thymidine and a minimum of 4 hr of in vitro exposure to 100 ng of ovine follicle-stimulating hormone (FSH) before a significant increase in DNA synthesis was elicited compared with 30-120 min for the Sasco breed. Peak responsiveness to FSH was observed at 8-hr incubation for the Harlan strain with significant increases in DNA per follicle at 8-12 hr. Both strains increased DNA synthesis with as little as 25 ng of ovine FSH and the response was elicited in all growing follicles, from preantral stages with one to four layers of granulosa cells, lacking theca (Stages 1-4) to mature antral follicles (Stages 8-10). A recombinant bovine FSH, devoid of luteinizing hormone activity, was not as effective as ovine FSH (which has 4% luteinizing hormone contamination) in stimulating DNA synthesis by large preantral and antral follicles. In vitro responsiveness to ovine FSH was abolished in the absence of Ca2+ in the culture medium and 0.05 mM Ca2+ was the optimal amount. For both strains of hamsters, the highest rate of DNA synthesis in response to endogenous gonadotropins was on the morning of estrus--when the second surge of FSH was in progress--and Harlan follicles in vitro also showed maximal stimulation by FSH on this day. Where the two strains differed was that the Harlan strain did not show an increase in follicular DNA synthesis on the afternoon of proestrus--when the preovulatory increase in gonadotropins commenced. When expressed as DNA per follicle, DNA approximately doubled from Stages 1 to 5 and then entered a new growth phase at Stage 6 (large preantral follicles) with a steeper increase. Collectively, these experiments show that strain characteristics can alter the latency and degree of follicular DNA replication in response to endogenous or exogenous FSH.     

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.     

Isolation and characterization of canine advanced preantral and early antral follicles

This study was designed to develop preantral follicle isolation and classification protocols for the domestic dog as a model for endangered canids. Ovary donors were grouped by age, size, breed purity, ovary weight and ovary status. Ovaries were randomly assigned to 1 of 3 digestion protocols: A) digestion and follicle isolation on the day of spaying; B) storage at 4 degrees C for 18 to 24 h prior to digestion and follicle isolation; C) digestion on the day of spaying, then incubation at 4 degrees C for 18 h prior to follicle isolation. Minced tissue was placed in a collagenase/DNase solution at 37 degrees C for 1 h. Follicles were classified by oocyte size and opaqueness and by size and appearance of the granulosa cell layers. Preantral follicles contained small, pale oocytes. Preantral follicles containing grown oocytes with dense cytoplasmic lipid were designated as advanced preantral. Only advanced preantral and early antral follicles were examined and classified further. Group 1 follicles had incomplete or absent granulosa layers, Group 2 follicles had several intact granulosa layers, while Group 3 were vesicular (early antral) follicles. Misshapen or pale grown oocytes were classified as degenerated. The percentage of intact germinal vesicles (GV) was recorded for each Group. Digestion Protocol B produced the lowest percentage of degenerated follicles (P < 0.01). Prepubertal donors had fewer (P < 0.01) follicles in each Group and more (P < 0.001) degenerated follicles than older bitches. Larger ovaries yielded the highest total number of follicles (P < 0.05). Ovary status did not affect follicle yield. Oocytes from Group 1 follicles had fewer intact GVs than those from Group 2 or Group 3 (P < 0.0001). These findings provide an opportunity for quantitative studies of the factors regulating folliculogenesis in the domestic dog as a model for endangered canids.     

Hormonal regulation of activities of 4-ene-5 beta- and 5 alpha-reductases and 17 beta-hydroxy-dehydrogenase in immature golden hamster testis

We have reported [1-3] in immature golden hamster testis that 5 beta-reductase is localized in the tubular nongerm cells, while 5 alpha-reductase is present in the interstitial tissue and that the 17 beta-hydroxy-dehydrogenase activity is found predominantly in the tubular nongerm cells. Hormonal regulation of these enzyme activities was examined in the present study. Male golden hamsters were hypophysectomized on day 22 after birth. The hypophysectomized hamsters in groups of 3-8 were injected daily with 10 micrograms NIH-LH-S19, 50 micrograms NIAMD-Rat-FSH-B-1, 8 or 16 micrograms NIAMD-oFSH-13, 8 micrograms NIAMD-oFSH-13 plus 5 or 10 micrograms NIH-LH-S19, 1 mg testosterone propionate or saline for 5 days starting from day 23. Testicular homogenates of the treated hamsters and intact hamsters on day 28 were incubated with [14C]4-androstene-3,17-dione and NADPH, and enzyme activity (nmol/testes/h) was estimated. The activities of 5 beta- and 5 alpha-reductases and 17 beta-hydroxy-dehydrogenase decreased significantly 6 days after hypophysectomy. In the hypophysectomized hamster testis, a distinct response to FSH but not to LH in the activities of 5 beta-reductase and 17 beta-hydroxy-dehydrogenase was found. The injection of LH in addition to FSH showed no significant additive effects on these enzyme activities. The 5 alpha-reductase activity was stimulated significantly by LH plus FSH but not by LH alone, FSH alone or androgen. These results show that 5 beta-reduction of 4-ene-3-ketosteroids takes place in the Sertoli cells under the influence of FSH while 5 alpha-reduction occurs in the interstitial cells under the influence of LH and FSH in immature hamster testis.