Longitudinal studies were performed in a colony of aging female rats, from 4-33 months of age, to determine the chronological change in reproductive patterns and the changes in sex steroid, prolactin and gonadotropin secretion associated with different reproductive states. The present study demonstrates that the incidence (65%) of irregular estrous cycles in aging rats increased abruptly from 10-12 months of age. Subsequently, female rats became chronically anovulatory with persistent vaginal cornifications and their ovaries contained developed follicles but no corpora lutea. The highest incidence (65%) of constant estrous (CE) rats occurred at the age of about 19 months. During the anovulatory state, CE rats displayed low to medium levels of serum estradiol, estrone, testosterone and androstenedione, low levels of progesterone and minimal levels of 20Î±-hydroxyprogesterone. Preovulatory increases in gonadotropin and prolactin release, similar to those seen in young cycling rats on proestrus, were not observed in CE rats. Whereas serum basal LH levels remained unaltered, morning FSH levels were increased in CE rats. The latter may account for the persistent follicular development in aging rats during chronic anovulatory state. Serum basal prolactin levels were normal in CE rats during the early phase (11-16 months of age) of the anovulatory state, but were subsequently increased 3 to 4-fold beyond 24 months of age. Moreover, ovariectomy at a young age prevented the increased pituitary prolactin release in old female rats. These results suggest that continuous exposure to medium levels of estrogens, particularly in the presence of sustained low progesterone secretion, may alter pituitary secretion of prolactin in aging rats. With further advance of age and following many months of anovulatory function, aging female rats exhibited ovulatory activity at irregular intervals. After each ovulation, formed corpora lutea were maintained for a prolonged period, presumably due in part to the existing high prolactin levels in the circulation of older female rats. These corpora lutea in old "pseudopregnant (PSP)" rats were functional as indicated by active secretion of progestins, with 20Î±-hydroxyprogesterone levels greater than those of progesterone in the circulation. These results indicate that the ovaries of aging rats retain their functional capacity to develop follicles and corpora lutea and to secrete steroid hormones. Although the cause(s) responsible for cessation of normal ovulatory cycles in aging female rats is unknown, the present study demonstrates that the chronic anovulatory state in aging female rats is characterized by significantly reduced ovarian secretion and the lack of cyclic increases in pituitary gonadotropin and prolactin release. The causal relationships between the decreased ovarian steroid production and the absence of preovulatory surges of gonadotropin release in aging CE rats remain to be determined.
The present study was undertaken to characterize hormonal and morphological events in 4 day and 5 day rat cycles, in order to test 3 different theories concerning the etiological factors altering follicular phase length in mammals. Three rats were killed at each 2 h interval throughout each cycle type. In addition to organ weight measurements, serum levels of estradiol, progesterone, 20Î±-hydroxypreg-4-en-3-one, LH, FSH and prolactin were assessed in terminal samples. The beginning of uterine intraluminal fluid accumulation coincided with the decline in progesterone levels seen during diestrus, rather than with the rise in estrogen and was delayed 12 h in the 5 day cycle from the time of last estrus. The retention of this fluid during the evening of proestrus was 6 h longer in the 4 day cycling rat. Ovulation occurred earlier in the 5 day cycling rats than in those with a 4 day cycle. No significant differences in the gonadotropins or 20Î±-hydroxypreg-4-en-3-one were seen when comparing the 2 types of cycles. Estradiol rose during the night of diestrus I (the same time in the cycle) and was identical in both cycle types. Progesterone was not released on proestrus prior to the LH surge in either type of cycle. However, we did find that progesterone values were higher for a longer period of time throughout diestrus I in 5 day cycles. These data have led us to conclude that the 5 day cycle is due to a prolonged progesterone secretion during the metestrous and diestrous stages of the cycle.
Male Holtzman rats were given a vitamin A deficient (VAD) diet from 21 days of age and retinoic acid after the onset of vitamin A deficiency. At 130 days of age, seminiferous tubules contain Sertoli cells, spermatogonia and preleptotene spermatocytes; serum LH and testosterone values are low normal, while FSH levels are high. Oral feeding of a single dose of 1 mg vitamin A (followed by a regular commercial rat pellet diet) causes reinitiation of spermatogenesis, although testosterone remains low and FSH does not return to normal for 60 days. After vitamin A treatment (PVA), kinetic characteristics based upon histologic criteria of the reinitiated spermatogenesis in VAD rats were normal. Pachytene spermatocytes can be seen by Day 14 PVA and spermatids by Day 24 PVA; elongation of spermatids was completed by Day 31 PVA and spermatozoa were formed by Day 41. Quantitatively, sperm production remained below normal at 150 days PVA, although epididymal sperm counts had continued to increase PVA to 50% of that in mature controls. The qualitative normalcy of spermatogenesis in PVA-VAD rats was demonstrated by fertility with normal litter size. Thus, VAD causes reversible germ cell depletion. Reinitated spermatogenesis in the VAD rat provides a kinetically normal, in vivo system in which functionally normal spermatozoa are produced and in which it may be possible to study the biochemical and morphological events of specific stages of spermatogenesis.
Chemical treatments previously shown to disrupt gap junctions were applied to rat ovaries with antral follicles prior to expressing the granulosa cells with a blunt spatula. Exposure of the ovaries to 6.8 mM EGTA [ethyleneglycol-bis-Î²-aminoethyl ether)-N,Nâ-tetracetic acid] and 0.5 M sucrose either by perfusion in situ or incubation in vitro generated monodisperse suspensions of granulosa cells with improved integrity as evaluated by several criteria. Similar numbers of granulosa cells (7-8 x 10 6 per ovary) were obtained either by direct physical expression or by pretreatment of the ovaries followed by physical expression. However, the ability of the granulosa cells to exclude trypan blue dye was consistently improved 2-3-fold by pretreatment with EGTA and hypertonic sucrose (from 25% to 40-80%). Likewise, in vitro synthetic capacities for protein, RNA and DNA were enhanced 2-6-fold, 5-10-fold and 10-20-fold, respectively. A qualitative one-to-one correspondence between protein synthesis and vital dye exclusion was demonstrable but the quantitative relationship between dye exclusion and macromolecular precursor incorporation appeared to be nonlinear. Cells obtained using the chemical pretreatments also demonstrated better survival in minimal medium for at least the first 12 h of culture. The enhancements observed could not be obtained by reversing the order of the chemical treatments or by treating granulosa cells after physical removal from ovarian follicles. Pretreatment of ovaries with EGTA and hypertonic sucrose appears to be a reliable procedure for improving the yield of monodisperse, viable, biochemically intact granulosa cells for use in in vitro examinations of follicular physiology and function.
This investigation was undertaken to explore further the dependence of the initial segments of the epididymis upon substances in the testicular fluid. The experimental groups consisted of rats castrated, rats with ligated ductuli efferentes and sham operated controls. The epdidymides were examined by light and electron microscopy 3 and 6 weeks postoperatively. The initial segments of the duct in both ligated and castrated animals exhibited 1) striking decrease in duct diameter and epithelial height, 2) a marked decrease in endoplasmic reticulum in the apical cytoplasm, 3) evidence of diminished activity of the Golgi complex, but 4) persistence of fluid uptake by pinocytosis. Daily administration of 500 Âµg testosterone proprionate beginning on the first postoperative day maintained the accessory gland weights at normal or supranormal values, but was ineffective in preventing the cytological regression of the initial segments of the caput epididymidis. In an effort to achieve levels of androgen in the blood comparable to those normally found in the rete testis fluid, castrated and ligated rats were implanted s.c. with 30 cm of Silastic tubing containing testosterone, each estimated to release 4 mg/day (Berndtson et al., 1974). Plasma testosterone levels when the rats were killed were 8-10 times normal. Despite these high levels of circulating androgen, cytological dedifferentiation of the initial segments was not prevented. More distal segments of the duct were essentially normal in dimensions and appearance. It is concluded that the initial segments of the rat epididymis require high intraluminal concentrations of androgen bound to androgen binding protein. The possibility that they are dependent upon some other, as yet unidentified, constituent of testicular fluid is not ruled out.
Explants of endometrium from the porcine uterus secrete labeled proteins into the growth medium when incubated in vitro in modified Eagleâs MEM 4 containing radioactive L-leucine. Tissue from 60 day pregnant animals secrete about 30 times as much radioactive material into the medium in 24 h as explants taken from nonpregnant animals on Day 3 of the estrous cycle. The secretions by tissue from pregnant animals were also qualitatively distinct and contained appreciable quantities of 2 basic proteins, lysozyme and a uterine-specific phosphatase. The latter was identical to an iron-containing purple protein previously purified either from uterine secretions of progesterone treated, nonpregnant animals or from allantoic fluid. This protein is believed to be involved in iron transport from mother to fetus. Explants from the endometrium of animals on Days 30, 45, 60, 75, 90 and 105 of pregnancy showed considerable differences in their capacities to produce the purple protein. Production was maximal by Day 60 tissue (2 mg/g tissue/24 h), but very low in tissue from late pregnancy (Day 105), an observation which may have considerable implication with regard to dietary iron supplementation of the pregnant sow. Neither the presence of progesterone nor estradiol-17Î² in the incubation medium altered the quantity or quality of the proteins produced by the explants during the 24 h incubation. We conclude that it is the hormonal environment of the endometrium at the time of surgery which primarily governs the quantity and quality of the proteins by short term cultured explants.
The follicular components responsible for the synthesis of progesterone (P), testosterone (T) and estrogen (E) have been identified by studying in vitro the steroidogenic pattern of theca or granulosa cells incubated alone with or without ovine FSH, ovine LH, P or T or incubated together with or without ovine LH. Granulosa cells, but not theca cells, isolated from 3 largest preovulatory and postovulatory follicles produced more P in response to the addition of exogenous gonadotropins. Among the 4 different types of follicles studied, this response of granulosa cells isolated from the largest follicle (F 1 ), the one destined to ovulate next, was the greatest. Both theca and granulosa cells isolated from preovulatory follicles produced 2-3 times more T when incubated in the presence of gonadotropins. Only theca cells from the second (F 2 ) and third (F 3 ) largest follicles synthesize more E when incubated with gonadotropins. The response of F 3 was greater than that of F 2 . Addition of T to the incubation system elicited E synthesis by theca cells of F 2 or F 3 , but not by granulosa cells of any size follicles. Again, the response of theca cells from F 3 was greater than that of F 2 . Furthermore, the ability of theca cells of F 3 , isolated at or shortly after the preovulatory in vivo LH surge, to aromatize T to E was significantly lower than that isolated several hours before the LH surge. Combination of the 2 follicular cell types from F 3 decreased P, but increased T (9- to 17-fold increase) and E synthesis. Theca cells incubated with exogenous P produced large amounts of T. We concluded that the cellular source for P is the granulosa cell and the source for T and E is the theca cell. Theca cells alone cannot synthesize T or E unless the precursor, P or T, respectively, is added. Both theca and granulosa cells are required for synthesis of T, followed by E. Thus, a 2 cell type model for synthesis of these 2 steroids is proposed. The present study also suggests that the steroidogenic potential of the follicular cells changes during the process of follicular maturation.
Within 7 days of the surgical induction of cryptorchidism, the Sertoli cells demonstrated an accumulation of lipid inclusions and dilatations of smooth endoplasmic reticulum. Aggregations of large vacuoles were observed at the basal aspects of the Sertoli cells and appeared to arise from local dilatations of the intercellular spaces between opposing inter-Sertoli cell junctions. These modifications of the inter-Sertoli cell junctional complexes disappeared as the cryptorchid state persisted, though some observations suggest that the associated membranes form complexly arranged bodies. The function of the Sertoli cells was altered in the cryptorchid testis as demonstrated by severe reduction in androgen binding protein (ABP) production by the 4 week cryptorchid testis and the lack of measureable ABP within the caput epididymidis. Serum FSH and LH levels became significantly elevated within 14 days of establishing cryptorchidism, suggesting diminished feedback from the damaged testis. Continuation of cryptorchidism was associated with progressive widening and folding of the peritubular tissue of the seminiferous tubule leading to bizarre arrangements of the tunica propria. The results are consistent with the proposal that in association with degeneration of the germ cells of the cryptorchid testis, the structure and function of the Sertoli cells are acutely sensitive to the raised intra-abdominal temperature.
Reproductive disorders in Se-deficient male rats from Se-adequate dams are described. After 4, 11 and 12 months on the Se-deficient (<0.02 ppm) diet, small, but sometimes significant, reductions in body and testicular weights were observed. Sperm morphology and motility appeared to be normal after the 4 month interval, but 50% of the animals in the 11 and 12 month interval groups produced sperm with impaired motility and a characteristic midpiece breakage. Because the morphologic anomaly does not appear to be correlated with effects on body and testicular weights, the effect of selenium on sperm is thought to be rather specific. The possible involvement of the Se-dependent enzyme glutathione peroxidase (GSH-P x ) in maintaining the integrity of the sperm membrane is discussed.
A study was done of the influence of length of photoperiod (control vs 16 h fixed photoperiod) and season (month) on: 1) changes in concentrations of plasma gonadotropins in ovariectomized (OVX) mares and 2) changes in plasma concentrations of gonadotropins and diameters of ovarian follicles preceding the onset of the ovulatory season in ovarian intact mares. In ovarian intact groups, intervals from start of project (January 6) to the first day of the ovulatory estrus, first appearance of a 30 mm follicle and onset of the ovulatory season (first ovulation) were shorter for the light-treated than for the control group. The changes in gonadotropin concentrations and follicular development preceding the onset of the ovulatory season (Day 0 = day of first ovulation) were not significantly different between light-treated and control groups. Mean plasma FSH levels fluctuated between Day -60 and Day -20 and then declined reaching a significant drop by Day -8. Mean plasma LH levels remained low until Day -8 and then increased reaching a maximum on the last day examined. The mean number of follicles 15-25 mm appeared to increase gradually from Day -60 to Day -20, remained elevated between Day -20 and Day -8 and then declined rapidly to a minimum on Day -1. The mean diameter of the largest follicle appeared to increase gradually from Day -60 to Day -8 and then increased rapidly between Day -8 and Day -1. The decline in FSH appeared to precede the decline in number of follicles 15-25 mm and the rise in LH. The rise in LH was temporally associated with the final growth and maturation of the preovulatory follicle. The ovulatory LH profile at the onset of the ovulatory season was similar in shape but lower in magnitude than the ovulatory LH profile during the middle of the ovulatory season. Removal of the ovaries during the anovulatory season did not evoke a postovariectomy rise in gonadotropin levels. However, there was a seasonal pattern of LH and FSH in ovariectomized mares and the LH levels increased approximately 2 months earlier in the light-treated group than in the control group. The rise in LH in the OVX groups occurred at approximately the same time as the onset of the ovulatory season in the intact groups.
Peripheral levels of estradiol, testosterone and progesterone were characterized during pregnancy in strains of mice which differ in prenatal survival following genetic selection. Strain variation in the secretory pattern of these hormones was minimal during the period that requires pituitary support of luteal function. In every line examined, a midpregnancy increase in circulating testosterone occurred, followed by a second increase in testosterone late in gestation. Estradiol levels also increased during the latter half of pregnancy, except in a line selected for rapid growth. In general, progesterone levels increased from midpregnancy until shortly before parturition. Dramatic strain differences in hormone levels and their secretory pattern during the second half of pregnancy suggest that placental-ovarian function has changed in response to selection more than has pituitary control over ovarian steroidogenesis.
Experiments were designed to examine the effects of exogenous testosterone on preantral follicles in the ovaries of estrogen-primed, hypophysectomized immature, female rats (HIFR). Parameters examined included ovarian weight, follicular morphology and intraovarian progesterone concentrations. Results show dose- and time-related decrements in ovarian weight and stimulation of follicular atresia during 4 days of treatment with testosterone at doses between 1 Âµg and 1 mg/day. The relationship between ovarian weight and logarithmic increments in the daily dose of testosterone was shown to be completely linear (correlation coefficient = -0.999, P<0.001). A similar relationship prevailed between ovarian weight and duration of testosterone treatment (1 mg/day) (correlation coefficient = -0.998, P<0.005). Ovarian weight reduction was directly related to concomitant stimulation of follicular atresia and was apparent within 24-48 h of initiating treatment with testosterone (1 mg). The effects of androgen on estrogen-primed ovaries were quantitatively and qualitatively reproduced by withdrawal of estrogen (in the absence of androgen) suggesting an antiestrogenic action of testosterone. Follicular atresia induced by testosterone or by withdrawal of estrogen was associated with a striking degree of thecal hypertrophy and evidence for the contribution of the theca of atretic follicles to the ovarian interstitial tissue was adduced. Since intraovarian concentrations of progesterone were not altered by treatment with exogenous testosterone, it is concluded that the atretic action of androgen represents its primary action on estrogen-stimulated preantral follicles in the absence of gonadotropic support.
Uterine blood flow (UBF) of 6 sows was monitored throughout an estrous cycle and the first 30 days of pregnancy using electromagnetic blood flow probes. Probes were placed around both uterine arteries of each sow on Day 12, 13 or 14 of the estrous cycle (first day of estrus = Day 0). The UBF (ml/min) was recorded twice daily for 15 min at 0700 and 1600 h. Values were averaged over both 15 min periods on each day for each artery and considered an estimate of UBF. Blood flow to uteri of nonpregnant sows was highest from Day -5 to estrus and this was followed by a gradual decline during the remainder of the estrous cycle. The pattern of blood flow to uteri of the sows during early pregnancy was similar to that observed during the previous estrous cycle until Day 11 after mating. On Days 12 and 13 of pregnancy, UBF increased (P<0.0l) 3-fold to 4-fold. No corresponding increase in UBF was observed in nonpregnant sows during this 2 day period. By Day 14 of pregnancy, UBF had returned to a level slightly higher than that observed on Day 11, remained relatively constant from Day 14 to 19, then increased (P<0.01) dramatically to Day 30. A second study was conducted using 7 sows made unilaterally pregnant on Day 2 postmating (first day of mating = Day 0). Fertilized ova were flushed from one oviduct and the ipsilateral uterine horn was ligated at the uterine body with silk sutures (nongravid horn). Blood flow probes were placed around both uterine arteries and UBF was recorded twice daily. Sows were slaughtered on Day 16 postmating. Five of the 7 sows had embryos and/or embryonic tissue in the nonligated uterine horn (gravid horn) at slaughter. Sows in which embryonic tissue was found had higher (P<0.05) blood flow to the gravid compared with the nongravid uterine horn on Days 12 and 13 of pregnancy. Corpora lutea on the ovary ipsilateral to a gravid uterine horn had higher (P<0.05) progesterone concentrations (Â±SEM) than did corpora lutea on the contralateral ovary (5.45 Â± 1.69 vs 2.07 Â± 0.49 Âµg/g luteal tissue).
The ultrastructural features of the intertubular tissue of the adult rat testis have been studied following the induction of cryptorchidism by surgical translocation of the testes into the abdominal cavity. The principles of stereology were used to assess the changes in the cytological features of the Leydig cells that occur 4 weeks after inducing cryptorchidism. Following the induction of cryptorchidism the principal changes observed were the hypertrophy of the Leydig cells with respect to cellular size and the increased quantities of Leydig cell organelles, particularly the mitochondria, Golgi membranes and the smooth endoplasmic reticulum. Despite the increase in organelles associated with steroid biosynthesis, serum testosterone levels of cryptorchid animals were either significantly lower or remained unchanged from normal control values, although serum LH levels were consistently elevated following induction of cryptorchidism. Furthermore, stimulation of the cryptorchid testis with hCG resulted in a subnormal increase in serum testosterone levels. The cytological changes in the Leydig cells are suggestive of increased steroid secretion and the normal or lowered levels of testosterone in the peripheral circulation of cryptorchid rats indicate the possibility of a biosynthetic block of testosterone production or alternatively the conversion of testosterone to other metabolites by the seminiferous tubule compartment.
The LH and FSH receptor content in thecal and granulosa cells of large antral follicles was measured by in vitro binding assays and in vivo autoradiography during the rat estrous cycle. Specific binding of [ 125 I]-hCG to granulosa cells increased from <1000 cpm/Âµg DNA on estrus, metestrus and diestrus to 6000 CPM/Âµg DNA on proestrus. Specific binding of [ 125 I]-hFSH to granulosa cells was consistently between 2000 and 3000 cpm/Âµg DNA throughout the cycle. Specific hCG binding to thecal cells also increased between diestrus and proestrus, while specific hFSH binding to thecal cells was essentially nondetectable. The increase in LH receptors in large antral follicles from diestrus to proestrus could be correlated with an increased ability of these follicles to produce cyclic AMP and estradiol during in vitro incubations with increasing concentrations (0.06-60 Âµg/ml) of oLH. Incubation of antral follicles collected on metestrus, diestrus and proestrus with and without testosterone revealed a progressive increase in both follicular aromatase activity and endogenous androgen production. It is concluded that in the presence of low and unchanging LH and FSH concentrations from metestrus to proestrus large antral follicles become more responsive to LH. This responsiveness is associated with an increase in LH receptors both in thecal and granulosa cells and increased production of estradiol. Although the hormonal stimulus for the increase in granulosa cell LH receptor on proestrus is not known, it is suggested that increased follicular estradiol might play a pivotal role.