A method for obtaining highly purified fractions of rat testicular cells is described. Single cell suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in pachytene primary spermatocytes, early spermatids, and cytoplasts detached from late spermatids were obtained. These fractions were further separated by equilibrium density centrifugation on gradients of Percoll. In this manner fractions of 3 x 10 7 pachytene spermatocytes (98% purity), 1.1 x 10 8 early spermatids (93% purity), and 1.1 x 10 8 cytoplasts (98% purity) were obtained within 6 h after sacrificing the rats. The cells appeared to be morphologically intact and to have retained their biochemical integrity. Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that histone 4 is synthesized during the pachytene stage, and confirmed that testis-specific histones are also synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing presumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.
The present experiments were carried out to characterize LH release during periods of low level LH secretion in the rat 4-day estrous cycle. Unanesthetized animals with jugular cannulae were bled on the morning (0930-1230 h) or afternoon (1330-1630 h) of either estrus, diestrus 1 (i.e., metestrus), or diestrus 2, or on proestrus morning (75 Âµl whole blood/6 min). The percent coefficients of variation obtained for alterations in blood LH levels during the morning or afternoon of each of the four stages of the estrous cycle, or when morning and afternoon values for each cycle stage were combined, were all significantly greater than intra-assay variation, indicating that LH release was pulsatile during each stage of the cycle. No significant morning vs afternoon differences in mean blood LH levels, LH pulse amplitude, or pulse frequency existed in any cycle stage. As the cycle progressed, changes occurred in mean blood LH levels which were due to changes in the characteristics of pulsatile LH secretion on each day of the cycle. On estrus, blood LH levels are the lowest of the entire cycle due to the occurrence of the slowest frequency of pulsatile release in the cycle (1 pulse/2 h). On the next day, diestrus 1, blood LH levels are elevated, and are the highest of all the periods of low level LH release in the cycle. This increase in blood LH levels from estrus to diestrus 1 is due to a marked increase in LH pulse amplitude (15 vs 38 ng/ml) as well as a shortening of the LH interpulse interval to 60 min. While this circhoral rhythm remained the same through diestrus 2 (55 min) and proestrus morning (63 min), the LH pulse amplitude decreased on these days to 20 and 16 ng/ml, respectively (compared with 38 ng/ml on diestrus 1), and therefore the mean blood level of LH on both days was significantly lower than on diestrus 1, although still higher than on estrus because of different pulse frequencies. Therefore, LH release is pulsatile during periods of low level LH secretion in the rat estrous cycle, and changes in the pulsatile characteristics of this release occur with different stages of the cycle.
A sensitive and highly specific radioimmunoassay procedure has been developed for measuring levels of circulating turkey prolactin. Mean plasma prolactin levels in broody turkey hens were found to be 9 times those of laying hens and 23 to 50 times those of molting hens, male poults, and adult toms. The plasma prolactin concentration of laying hens was found to increase gradually beginning about 10 days prior to the onset of broodiness, and to increase sharply at about the time of persistent nesting. Interrruption of incubation behavior by nest-deprivation and constant light was associated with a precipitous decline in prolactin levels. A hen that continued to exhibit incubation behavior despite this treatment showed no decline in plasma prolactin levels. A hen that continued to exhibit incubation behavior despite this treatment showed no decline in plasma prolactin concentration. These results confirm and expand the findings of some other investigators that incubation behavior in the turkey is associated with dramatically elevated prolactin levels.
The ovarian secretion of estradiol, androstenedione, testosterone, and progesterone and the concentration of LH, FSH, and prolactin were measured in the periovulatory period of five ewes with ovarian or utero-ovarian autotransplants. Samples of jugular and ovarian venous blood were collected every 1 or 2 h before and for 96 h after induction of luteal regression on Day 10 of the cycle by injection of 100 Âµg cloprostenol. Progesterone concentration in jugular vein plasma fell to less than 1 ng/ml in all ewes by 24 h, and coincidental to this decline there was a significant increase in the secretion of LH and prolactin and an associated rise in the ovarian venous concentration of estradiol, testosterone, and androstenedione. The rise in LH secretion was associated with a twofold increase in the frequency of episodic pulses each of which stimulated an increase in the secretion of estradiol. In contrast the concentration of FSH declined significantly in the 48 h after luteal regression. Shortly after the onset of estrus (48 Â± 2.5 h), there was a marked rise in the concentration of prolactin, FSH, and LH which reached a peak at 61 Â± 4 h. At the start of this preovulating LH surge, there was a further substantial stimulation in the secretion of estradiol, testosterone, and androstenedione, followed by a sharp fall within 3, 5, and 7 h, respectively. In the 24 h after the preovulatory surge, there was a marked decline in the concentration of LH and prolactin as well as all ovarian steroids. However, within 12 h there was a further rise in the concentration of FSH which reached a second peak at 23 h after the LH peak. These results suggest that the increased secretion of estradiol from the preovulatory follicle is due to stimulation by episodic pulses of LH which occur with increased frequency as the concentration of progesterone falls during luteal regression. The sustained rise in LH which occurs during the preovulatory surge stimulates and then markedly inhibits aromatase activity and eventually all steroid secretion from the follicle. Thus the final stages of development of the preovulatory follicle are determined not by FSH but by the pattern of secretion of LH.
Cells with the ultrastructural features of macrophages were seen within corpora lutes of mice shortly after induction of ovulation with gonadotropins. Upon isolation, mononuclear phagocytes from luteal tissue had histochemical and functional properties characteristic of macrophages from other body sites. These attributes included intense staining for nonspecific esterase activity, phagocytosis of IgG-coated red blood cells via an Fc receptor, ingestion of latex beads, and trypsin-resistant adherence to the surface of culture dishes. Furthermore, the ovarian macrophages specifically bound to their surface a monoclonal antibody against a plasma membrane protein of mononuclear phagocytes. When mouse granulosa cells were co-cultured with peritoneal macrophages, progesterone secretion was increased, and the magnitude of the increased steroidogenesis was directly related to the number of mononuclear phagocytes added to the cultures. Likewise, cultured luteal cells from superovulated mice secreted more progestins in the presence of peritoneal macrophages. Luteal cells also produced more progesterone when they were recombined in culture with autologous macrophages from corpora lutea. The mononuclear phagocytes from ovarian tissue appeared to have a greater ability to stimulate luteal cells than did peritoneal macrophages. Conditioned media from cultured macrophages failed to stimulate steroid secretion by granulosa cells, suggesting that close proximity of the two cells is required for the effect on luteal cell function. Thus, it appears that macrophages within corpora lutea may play a role in maintaining progesterone secretion by luteal cells.
The present experiments were carried out to determine if LH release during the ovulatory LH surge in the rat was pulsatile. Unanesthetized rats with 4-day estrous cycles were bled through jugular vein cannulae at a rate of 10 Âµl whole blood/2-3 min from 1400 to 1930 h on proestrus during the ovulatory LH surge. Ovulation occurred in all rats that had surges of LH release. These surges consisted of ascending, plateau, and descending periods. LH release was pulsatile during the plateau portion as indicated by a significant increase in the percent coefficient of variation for individual bleeding periods when compared with assay variation, and during the ascending and descending phases as evidenced by pronounced nadir-to-peak changes in blood LH levels which were greater than the minimum significant change detected by radioimmunoassay. Blood LH levels increased during the ascending phase and then plateaued because of high amplitude LH pulses (538 ng/ml in the ascending phase and 572 ng/ml in the plateau phase), which occurred with LH interpulse intervals of 16 and 23 min, respectively. Blood LH levels declined during the descending phase since the pulse amplitude (386 ng/ml) significantly decreased, and in comparison with the ascending phase the LH interpulse interval lengthened significantly to 29 min. These studies indicate that pulsatile LH release occurs during the ovulatory LH surge on proestrus in the rat, and that the rise, plateau, and fall in blood LH levels can be accounted for by changes in the LH pulse amplitude and frequency during different phases of the surge.
Midday plasma concentrations of estradiol (E 2 ) and progesterone (P) were measured in aging C57BL/6J mice showing either prolonged estrous cycles or persistent vaginal cornification (PVC). In younger mice (5.5-7.5 months), the modal cycle length was 4 days, and the profiles of these hormones during the cycle were similar to those previously reported. By 10-12.5 months, the modal cycle length had increased to 5 days. Although midday levels of P were unimpaired at this age, the profile of E 2 was markedly altered. Proestrus (Day 1) and estrus (Day 2) levels of E 2 were indistinguishable from those of younger mice, but basal values on Day 3 and the preovulatory rise beginning on Day 4 were reduced in older mice by 80% and 45%, respectively. As a result, attainment of preovulatory levels of E 2 equivalent to those of younger mice was delayed by about 1 day, which corresponded to the average net increase in median cycle length at this age. In 14-month-old PVC mice, levels of E 2 were comparable to the basal values of younger cycling mice and threefold greater than ovariectomized values. However, P levels were 50% lower than basal values in cycling mice and indistinguishable from values in ovariectomized mice. Consequently, the E 2 :P ratio was elevated nearly twofold in PVC mice. Because transient experimental suppression of the preovulatory rise of E 2 lengthens estrous cycles in young rodents, we hypothesize that the delayed rise of E 2 in older mice contributes to their prolonged cycles. In addition, the increased E 2 :P ratio in PVC mice creates a relatively unopposed estrogenic milieu which may accelerate the development of the pituitary tumors and hypothalamic dysfunction that characterize aged female mice.
Overall rates of glucose utilization at physiological concentrations (5.5 mM) by primary cultures of rat Sertoli cells have been estimated to be 445 nmol/mg cell protein/h when determined by rates of incorporation of 5-[ 3 H]-glucose into [ 3 H]-H 2 0, and 486 nmol/mg protein/h when determined by rates of incorporation of U-[ 14 C]-glucose into labeled products (CO 2 , anions, lipids, and glycogen). Of total U-[ 14 C]-glucose utilized, 2.9% was converted to [ 14 C]-C0 2 ; 95.8% was converted to [ 14 C]-anions, most of which could be accounted for as lactate; and the remainder was incorporated into lipids and glycogen. About half the [ 14 C]-CO 2 formed from labeled glucose was calculated to be derived from oxidation via the pentose phosphate pathway. In the presence of the artificial electron acceptor phenazine methosulfate, the rate of conversion of 1-[ l4 C]-glucose to [ 14 C]-CO 2 was increased over 10-fold, whereas the incorporation of 6-[ 14 C]-glucose into [ 14 C]-CO 2 was not changed. Under these conditions, over 33% of total glucose utilized by Sertoli cells could be accounted for by the pentose phosphate pathway. However, this potential was not utilized in the absence of the artificial electron acceptor. Neither the overall rates of glucose utilization nor the metabolic fate of glucose was appreciably influenced by treatment of cells with follicle stimulating hormone, insulin, or dibutyryl cyclic AMP under any conditions investigated. The possible physiological importance of the high rate of lactate production by Sertoli cells was considered in relation to the role of lactate as a substrate for germinal cells in the adluminal compartment of the seminiferous tubule.
Protein synthesis and secretion by the epididymis was studied in vitro by incubating tissue pieces with radioactive amino acids or one of a variety of radioactive sugars. When amino acids were used as precursors, about 15% of the incorporated radioactivity was released into the medium; the release was 36-65% in the case of sugar precursors. In different regions of the epididymis, pronounced variations were apparent in the profile of secreted proteins formed from amino acids or from galactose, mannose, or fucose. Total protein synthesis as measured by [ 35 S) methionine incorporation was decreased by â¼30% following castration of animals, and the proportion of incorporated radioactivity which was secreted was reduced from 15% to 10%. Only a few qualitative changes were evident in the types of protein secreted. Compared with general protein synthesis, the incorporation of sugars into glycoproteins was affected by castration to a much greater extent, with mannose incorporation being reduced by up to 90% in the cauda. Protease inhibitors (TLCK, TPCK, PMSF, benzamidine, phenanthroline) reduced total protein synthesis from [ 35 S] methionine but did not alter the profile of secreted proteins. Procaine also reduced total protein synthesis, and in the caput, but not the cauda, it caused a marked change in the pattern of secreted proteins. This differential effect of procaine did not appear to be due to an effect on "signal peptidase." Tunicamycin had little effect on protein synthesis and secretion by the epididymis when [ 35 S] methionine was used as the precursor, although it did cause pronounced changes in protein secretion by testicular tissue. Greater effects of tunicamycin were evident when radioactive sugars were used as precursors, particularly in the case of [ 3 H] mannose. The incorporation of this precursor was reduced to 69% and 49% of normal in the caput and cauda, respectively. However, tunicamycin did not alter the profile of secreted radioactive proteins resulting from any of the incubations with radioactive sugars.
The Leydig cell hypertrophy and loss of LH receptors that accompanies the surgical induction of bilateral cryptorchidism has been attributed to elevated serum LH levels. This study demonstrates that in unilaterally cryptorchid rats, the Leydig cell hypertrophy and loss of LH receptors is confined to the abdominal testis and there is no significant change in these parameters in the scrotal testis. These results and the failure of serum LH levels to increase following unilateral cryptorchidism until 4 weeks, indicate that local factors within the cryptorchid testis must result in the changes in Leydig cell function. In addition to the loss of LU receptors and Leydig cell hypertrophy, the cryptorchid testis showed a hyperresponsive testosterone response to hCG stimulation in vitro and a loss of FSH receptors whereas both of these parameters were normal in scrotal testes of the unilaterally cryptorchid rats.
The carcinogenic polycyclic hydrocarbon benzo(a)pyrene(BP) is a ubiquitous urban pollutant and is a component of cigarette smoke. Studies reported here were designed to investigate the effect of daily oral doses of 0, 10, 40, and 160 mg BP/kg maternal BW on Days 7-16 of gestation on pregnancy maintenance, and on fetal development and survival of CD-1 mice. Postnatal development and reproductive function also were investigated in F 1 male and female mice exposed to BP in utero. Total sterility was observed in 97% of F 1 mice exposed prenatally to 40 or 160 mg BP/kg. Fertility was markedly impaired in F 1 animals exposed in utero to 10 mg BP/kg. After 6 months on a breeding study, female mice exposed to this dose of BP in utero gave birth to significantly fewer and smaller litters. Male mice exposed to 10 mg BP/kg impregnated 35% fewer females than did control males. The impaired fertility in F 1 , mice exposed to BP in utero was associated with marked alterations in gametogenesis and folliculogenesis and a dramatic decrease in the size of the gonads. Germ aplasia was observed in males as well as a reduction in the size of the seminiferous tubules and an increase in the quantity of interstitial tissue. Microscopic examination of ovarian tissue from F 1 females exposed to 10 mg BP/kg revealed marked hypoplasia with very few corpora lutea or follicles. The data demonstrate a sensitivity of fetal gonads to BP which is similar to effects observed with another polycyclic aromatic hydrocarbon, 9, 10-dimethyl-1,2-benzanthracene (DMBA).
Attempts were made to characterize the time courses of concentrations of circulating LH, FSH, PRL, testosterone (T), and cortisol throughout the light-dark cycle (lights on 0600-1800 h) in intact adult male rhesus monkeys. Blood samples were withdrawn from five animals every 20 min with a remote sampling device that permitted continuous access to the venous circulation with minimal restraint of the animal. Mean concentrations of plasma T were highest during darkness and declined abruptly shortly after the lights came on to reach a nadir â¼3 h later at 0900 h. Mean T levels then increased progressively and in an apparently rhythmic fluctuating fashion throughout the remainder of the light phase of the 24 h cycle. Individual animals exhibited very striking moment-to-moment changes in plasma T concentration composed of 7-12 discrete peaks during the 24 h cycle. The frequency of these episodes of testicular T secretion throughout the light phase of the cycle, and particularly during the initial 6 h of illumination (approximately one plasma T peak every 5 h), was noticeably slower than that during darkness (approximately one peak every 2 h). The amplitude of plasma T peaks, however, was similar throughout the entire light-dark cycle. Essentially every peak in concentration of circulating T was preceded by a brief, but distinct, elevation in plasma LH, and most discharges of LH (89%) were followed by an episode of T secretion. These findings suggest that in the male rhesus monkey the characteristic diurnal variation in circulating T is determined primarily by changes in the frequency of episodic testicular T secretion which, in turn, appears to be occasioned by a corresponding pattern of intermittent release of LH. Plasma prolactin levels were lowest at â¼0800 h, and concentrations of circulating cortisol exhibited, as expected, a diurnal variation typical of this plasma steroid. The time course of FSH was not forthcoming because concentrations of this plasma gonadotropin in the circulation were usually immeasureable by the RIA employed.
We examined the effects of physiological concentrations of estradiol (E 2 ) and progesterone (P) on plasma LH, FSH, and prolactin (PRL) and on LHRH concentrations in several microdissected brain regions in ovariectomized (OVX) rats. One week after ovariectomy (Day 0), rats received Silastic capsules containing only sesame oil or 37.5, 75.0 or 150 Âµg E 2 /ml of oil s.c. The E 2 capsules produced plasma estrogen concentrations of 7.0, 9.6, or 15.4 pg/ml, respectively, on Day 2 whereas oil-treated controls had 5.4 pg/ml of E 2 in plasma. All three E 2 -treated groups of rats had LH surges of comparable magnitude during the afternoon of Day 2. Two Silastic capsules of P (50 mg/ml) were implanted at 0900 h on Day 2 into E 2 -treated rats. In animals in which E 2 levels were 7.0 or 9.6 pg/ml, P only moderately amplified the LH surges. However, when plasma E 2 concentrations reached 15 pg/ml, P treatment evoked a massive release of LH and advanced the time of release by 1 h. In contrast, the stress of inserting oil capsules into ether-anesthetized E 2 -treated rats at 0900 h on Day 2 had no effect on afternoon LH surges in these animals. FSH surges occurred only in rats receiving both E 2 and P. PRL surges were induced using the highest concentrations of E 2 and were advanced in time by P. When the highest dose of E 2 was used, LHRH concentrations in the median eminence (ME) increased prior to and decreased during the E 2 P-induced surge. These changes were not paralleled by any changes in the other anterior brain regions we examined (suprachiasmatic preoptic nucleus, suprachiasmatic nucleus, medial preoptic nucleus, anterior hypothalamic nucleus, and retrochiasmatic area). No changes in LHRH concentrations were observed under any other steroid treatment regimen. Higher plasma E 2 concentrations induced by the 150Âµ g/ml E 2 capsule were correlated with higher LHRH concentraions in the median eminence concomitant with a parallel trend in some of the anterior brain areas: MPN (P<0.06), SPN (P<0.06), and RCA (P<0.03).
Age-related changes in the Leydig cell population of horses between 2 and 20 years old were characterized by a twofold increase in Leydig cell number per gram parenchyma, a threefold increase in Leydig cell number per testis, a threefold increase in Leydig cell volume per gram parenchyma, and a fivefold increase in Leydig cell volume per testis. Due to the increased volume of Leydig cells per gram of testicular parenchyma and the increased size and accumulation of lipofuscin granules in individual cells, the gross appearance of the parenchyma became darker with age. The Leydig cell population increased with age by replacing the noncellular components of the interstitium and did not alter the proportion of the testis occupied by blood vessels, lymph vessels, or seminiferous tubules. Both the diameter and length of seminiferous tubules increased with age. Daily sperm production per testis increased between postpuberty and adulthood yet did not increase significantly beyond adulthood. However, the number of Leydig cells per testis continued to increase in the aging horse. Other significant changes included increases in testicular weight, volume of Leydig cells per testis, seminiferous tubular length per testis, and daily sperm production per testis in April-May compared with February-March.
Reproductive behavior and ovarian and endocrine relationships were studied in the cat during and following a controlled mating regimen during estrus (n = 12) and an estrous period without mating (n = 5). Ovulation was confirmed laparoscopically only in animals experiencing coitus. Average (Â± SEM) duration of estrus and follicle numbers were similar (P>0.05) in ovulating (5.8 Â± 0.2 days; 5.0 Â± 0.5 follicles) and nonovulating (6.4 Â± 0.6 days; 5.2 Â± 1.0 follicles) queens. Duration of the luteal and interestrous interval in ovulating cats was 38.2 Â± 2.8 and 50.3 Â± 2.7 days, respectively. Following repeated mating, 86.6% of vesicular follicles detected on Day 1 of estrus ovulated, resulting in a mean CL number of 4.3 Â± 0.5. Mean serum estradiol-17Î² concentration was variable but generally greater than 20 pg/ml during estrus in both mated and unmated queens. In the former group repeated matings elicited an LH response during the first 2 days of estrus (Days 1 and 2) in 10 of 12 queens. Two cats mated 3 times daily failed to produce an LH response until Day 2. On Day 1 mean LH rose from 4.3 ng/ml (0 h) to 50.4, 74.1, and 25.8 ng/ml at 4, 8, and 14 h, respectively. On Day 2, average LH changed from 10.3 ng/ml (0 h) to 27.7, 23.4, and 9.6 ng/ml at 4, 8, and 14 h, respectively. Generally no further increase in LH response was detected even though queens continued to mate. The initial rise in mean serum progesterone occurred on the morning of Day 4 or â¼64-68 h after the first detected LH peak. In five of 12 cases, ovulation was completed within 48-52 h of the LH peak. In the remaining seven cats, ovulation occurred 52 h or more after the LH peak but was completed in all animals by midafternoon on Day 5. Data from individual cats during estrus were similar to the average results; however, dramatic temporal fluctuations in steroid hormones, particularly estradiol-17Î² were often observed. Vesicular follicles were observed on the ovaries of individual cats during all stages of the luteal and interestrous interval. During these periods elevations in serum estradiol-17Î² above basal concentrations were occasionally detected particularly following the end of the luteal phase. During the latter phase, mean CL diameter increased gradually with elevations in serum progesterone; however, regression of visible luteal tissue was gradual with CL remnants remaining visible through the end of the interestrous interval. In unmated queens the mean serum estradiol-17Î² profile during and after estrus was similar to the ovulating group. LH and progesterone were sustained at basal concentrations throughout these periods. These data interrelate events of the reproductive cycle of the cat and suggest that 1) the mated estrual queen exhibits a gradual decline in pituitary LH response after repeated coitus; 2) "coitusto-ovulation" interval is an inappropriate parameter for this species since neither single nor multiple copulation always ensures an LH response and, thus, ovulation; 3) although follicular activity is greatest during estrus, periods of follicle growth and regression appear to occur continually even during the luteal phase.
Insulin is a requisite for the FSH-mediated induction of LH/hCG receptors in porcine granulosa cell monolayers maintained in serum-containing medium (May et al, 1980). In this report we describe further the role of insulin on this process and on other parameters of cell monolayer performance, e.g., plating efficiency, cell growth, basal and gonadotropin-stimulated progesterone production, and aromatase activity. Insulin did not affect the plating efficiency (>90%) of freshly harvested, viable granulosa cells. During the first 2 days of culture, monolayer cell content increased slightly under insulin-free conditions. Insulin did not alter this slow rate of cell division. After the first 2 days, however, cell and protein content of monolayers deprived of insulin decreased significantly (60%, P<0.001) relative to insulin-treated monolayers in which cell and protein content were either maintained or increased relative to the initial innoculum. Basal progesterone secretion declined with time in culture but was maintained or slightly increased under the influence of insulin. Both FSH- and hCG-stimulated progesterone production were significantly enhanced (P<0.001) by insulin after the first 2 days of culture. FSH-stimulated progesterone secretion was dose-dependent with respect to insulin as was FSH-mediated LH/hCG receptor induction. The immature granulosa cells used in these studies assumed a fibroblastic morphology in the absence of added hormones. Insulin induced an epithelioid morphology in vitro, characteristic of more mature cells. LH and FSH alone were incapable of altering the fibroblastic morphology and did not affect the change brought about by insulin. Thus, gross morphological maturation of granulosa cells in vitro correlated with insulin-mediated biochemical differentiation, e.g., LH/hCG receptor induction and steroidogenic capacity. Although insulin enhanced several markers of cell differentiation, it did not ameliorate the loss of aromatase activity during culture, a characteristic of this model system. During the first 2 days of culture and in the presence of testosterone, granulosa cell monolayers secreted significant amounts of estrogen. However, this capability declined rapidly with time in culture. Neither insulin alone nor insulin combined with FSH acutely stimulated aromatase activity, nor did these treatments protect against the loss of aromatase activity. Highly purified porcine relaxin and commercially available multiplication-stimulating activity (MSA), compounds possessing insulin-like structure and activity, respectively, were incapable of replacing insulin as a requisite for FSH-mediated LH/hCG receptor induction or gonadotropinstimulated progesterone secretion. Our results indicate that insulin is critical for the maintenance of several functional properties of porcine granulosa cell monolayers, and thus should be considered for routine use in studies of the regulation of ovarian function in vitro.
The interrelationship between follicular and luteal function during pregnancy in the rat was examined by in vivo and in vitro methods. Corpora lutea (CL) and nonluteal ovarian tissues (NLO) were removed on Days 2 to 22 (at 2 day intervals) (day sperm-positive = Day 1 of pregnancy), and incubated separately for 2 h to determine the production rate of steroids. Changes in progesterone (P), 20Î±-dihydroprogesterone (20Î±-OHP), testosterone (T), and estradiol-17Î² (E 2 ) in peripheral blood, CL, and NLO, and levels of LH and FSH in serum and pituitary were measured by radioimmunoassay. A marked decline in the in vitro production rate of E 2 and T in NLO occurred between Days 14 and 18 followed by an abrupt increase on Days 20 and 22. These changes correlated with the levels of serum LH between Days 14 and 22. Serum FSH also declined on Day 16 but returned to basal levels by Day 18. Despite the fall in NLO production of E 2 , serum E 2 started to increase on Day 14 with a progressive rise continuing until term. A marked increase in luteal content of E 2 occurred on Day 16 and continued until Day 22, whereas in vitro production of E 2 by CL declined from Day 14 onward. Serum T also began to increase on Day 14 with the peak attained between Days 18 and 20, paralleling the rising E 2 levels. In vitro production of luteal T increased on Day 12 with peak values present between Days 16 and 20, paralleling the pattern of serum T. Maximal values of serum P occurred between Days 14 and 16 and decreased after Day 18. In vitro production of luteal P increased abruptly on Day 4, with peak values on Day 10 and a decline by Day 16. There was a second peak in in vitro production of P on Day 22, although serum and luteal levels of P were already low. Changes in serum levels of 20Î±-OHP and its in vitro production rate were inversely related to changes in P throughout pregnancy. These findings indicate that at the end of pregnancy the CL produce large amounts of P in vitro after functional luteolysis has occurred in vivo. The high levels of P during midpregnancy may be involved in the suppression of follicular maturation, probably by lowering basal levels of serum LH. Furthermore, between Days 14 and 18, secretion of T and E 2 represents proportionally more luteal than follicular sources of the hormones in the pregnant rat.