BIOLOGY OF REPRODUCrION, 6-68 (197) The Distribution of Ovarian -$-Hyd roxysteroid Dehyd rogen ase Activity in the Golden Hamster During the Estrous Cycle, Pregnancy, and Lactation GORDON C. BLAHA AND WENDELL W. LEAVITT Departments of Anatomy and Physiology, University of cincinnati College of Medicine, Cincinnati, Ohio 19 Received July 1, 197 The distribution of the enzyme --hydroxysteroid dehydrogenase (HSD) was studied histochemically in sections of ovaries from normal golden hamsters. During the estrous cycle, the enzyme activity increased in new corpora lutea to a maximum at days after ovulation and then declined. The theca of growing follicles gradually developed activity during the cycle, attaining maximal activity on Day before ovulation when corpora lutea were inactive. Only on Day of the cycle did the granulosa show $HSD. The interstitium was histochemically active at all times. The corpora lutea of pregnancy developed strong enzyme activity by Day which was maintained until Day 1 and faded before delivery on Day 16. Growing follicles during pregnancy had activity in the theca on all days, but it became weaker on Day 16. The granulosa of follicles showed some HSD by Day of pregnancy, and this increased after Day 8 to become as strong as that in corpora lutea on Day 1. The strong 1iHSD activity in the granulosa persisted until after delivery. Follicles did not ovulate before or after delivery, and the corpora lutea and antral follicles of pregnancy disappeared during the first few days of lactation. By 8 days of lactation, only interstitium had HSD. We propose that the changes in the distribution and activity of fl- HSD in the ovary of the golden hamster reflect the changing ratios of gonadotropins and the resulting effects on ovarian steroid secretion. The enzyme --hydroxysteroid dehydrogenase ($HSD) is necessary for the synthesis of progesterone from pregn--en--ol- -one (pregnenolone). It was first localized histochemically by Wattenberg (198), using the principal of formazan precipitation from tetrazolium salts in areas of dehydrogenase activity. The identification of ovarian compartments capable of synthesizing progesterone has a direct bearing on reproductive 1This work was supported by Research Grants M68-19 and M-7--C from the Population Council. A preliminary report of this work has appeared (Anat. Rec. 166, 79, 197). functions in the hamster, since progesterone is essential for psychic estrus (Frank and Fraps, 19), implantation, and pregnancy (Prasad, Orsini, and Meyer, 196; Orsini and Psychoyos, 196). The localization of /HSD in the ovary does not necessarily signify progesterone secretion, but it does indicate a capacity for progesterone synthesis. The lack of flhsd activity should preclude progesterone production. The present study was an attempt to obtain knowledge of the changing patterns of,hsd activity during various times of normal ovarian function in the golden hamster. 6 Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18
OVARIAN /HSD IN THE HAMSTER 6 MATERIALS AND METHODS Normal female golden hamsters, -6 months old, were caged individually under conditions of controlled temperature (-C) and lighting (lights on to 1 hr EST). Teklad hamster chow and water were provided to choice, and lettuce was given three times weekly. The estrous cycle was followed by the postovulatory vaginal discharge for several consecutive -day cycles. Matings were in the early dark period several hours before ovulation and were verified by observing coitus and checking for spermatozoa after at least 1 mm of mating. Day I of the cycle and pregnancy is the day following ovulation. Animals were anesthetized with sodium pentobarbital (9 mg/loo g ip). Before removal of ovaries, blood samples were taken from most animals for analysis of progesterone content (Leavitt and Blaha, 1971). Ovaries from at least one animal in each group were removed without prior bleeding. Ovarian $HSD was studied at each time period indicated in Table 1. The ovaries were weighed and then frozen at - to -C, either directly onto cryostat cutting blocks or in prechilled glass tubes. Frozen sections 1 thick were placed on clean glass slides and kept frozen. Sections were extracted in cold acetone ( C) for 1 mm before incubation to remove lipid and substrate, then rinsed in two changes of cold Tris buffer ( C, ph 7.,. NI). Sections were incubated for 1,, or hr at 7 C in ml of medium (. M Tris buffer, ph 7.; 1 mg Nitro Blue Tetrazolium; mg NAD; mg pregnenolone dissolved in. ml acetone). All compounds were from Sigma Chemical Co., St. Louis. Two types of control incubations were carried out. In the first, medium without steroid substrate was used to verify the specificity of the reaction. The second was done to verify the presence of NADH diaphorase which is necessary for transferring hydrogen from the dehydrogenase reaction to tetrazolium for formazan precipitation. For diaphorase, tissues were incubated for 1 mm at 7 C in ml of. M Tris buffer (ph 7.) with mg NADH and 1 mg Nitro Blue Tetrazolium. After incubation, all sections were fixed in phosphatebuffered formalin (ph 7) for 1 mm, rinsed in distilled water, counterstained lightly with neutral red, rinsed, and mounted in glycerol. Enzyme activity was evaluated subjectively using a -S scale similar to one previously described (Pupkin et a!., 1966). A minimum of 18 sections were examined for each animal (three sections/slide, three slides/ovary, both ovaries used). Control incubations with no substrate had no activity () in all cases. Since ovarian interstitial cells always contained Interstitium No. of animals 6 8 S TABLE HISTOCHEMICAL ACTIVITY OF OVARIAN #{16}HSDIN THE GOLDEN HAMSTER DURING THE ESTROUS CYCLE, PREGNANCY, AND LACTATION (RATED Estrous Day cycle (proes - trus) (estrus) Pregnancy Day 6 8 1 1 1 1 16 Lactation Day 8 SUBJECTIVELY ON A - SCALE) Granulosa Theca - I -S - - - -1 - S -1 - -1 1- o -1-1 Corpora lutea - - 1- -1-1- -1 None present Modal values based on a minimum of 18 sections per animal. I Interstitial IHSD activity was arbitrarily set at. = no detectable activity. 9HSD activity and their intensity did not vary appreciably on different days, interstitial activity was arbitrarily set at. This provided a basis for judging the intensity of fihsd activity in other ovarian structures as it varied during the cycle, pregnancy, and lactation. Photographs were taken within 1 day of incubation. Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18
6 BLAHA AND LEAVITT RESULTS Ovarian interstitial cells contained,hsd activity at all times studied (Table 1), and there was little variation in the intensity of interstitial jhsd activity on different days. The ovaries from different animals on any one day reacted similarly. No HSD was found in ovarian connective tissue, blood vessels, or surface epithelium, although NADH-diaphorase was present in cells of these areas. Estrous Cycle (Figs. 1-7). At an estimated 1 hr after ovulation, the activity of 9HSD in newly forming corpora lutea was barely equal to that of the interstitium. Light activity also appeared in the theca interna of growing follicles, but granulosa cells had none. By Day, corpora lutea were well organized and showed intense 1HSD activity which made them stand out from the surrounding interstitium and follicles. Antral follicles were larger but their thecal layer showed little IHSD and granulosa cells still had none. Luteal HSD decreased during Day, so that by late in the day, corpora lutea had only about the same degree of activity as the interstitium. The expanding follicles had HSD in the theca interna of about equal intensity to the interstitium, and the granulosa still had no activity on Day. During the light period on Day (proestrus), the theca interna of large follicles developed more intense 1HSD activity, the granulosa began to show some activity, and luteal enzyme activity declined. During the early part of the dark period on Day (behavioral estrus), the corpus luteum had almost no activity at a time when the theca interna of preovulatory follicles still showed considerable IIHSD. Pregnancy (Figs. 8-11). The ovaries on the first day after mating had the same pattern of /HSD activity as on the first day after ovulation during the cycle. By the second day of pregnancy, the corpora lutea had become organized and showed more IHSD than other ovarian structures. The theca interna of growing follicles on Day showed activity of about equal intensity to that of the interstitium. The same relative amount of HSD activity persisted from Day to Day 1 in the above structures. However, corpora lutea and follicles grew during this interval. Growing follicles showed a notable increase of $HSD activity in the granulosa layer. Light $HSD was seen in the granulosa on Day, and this activity increased somewhat to Day 1. By Day 1, the activity of $HSD in granulosa cells of large follicles equalled that seen in corpora lutea. This persisted until Day 1, when enzyme activity began to decrease in corpora lutea. By Day 16 of pregnancy, with parturition imminent, /HSD activity was notably less in corpora lutea than in interstitium, leaving the granulosa of follicles with the most enzyme activity. Lactation (Figs. 1-1). After nearly a day of lactation, the corpora lutea of pregnancy had no 19HSD activity. The follicles did not ovulate and underwent atresia. As lactation continued, only the interstitium had /HSD, and there were no antral follicles or corpora lutea. This ovarian condition persisted until the young were removed. The atrophic uterine condition observed in lactating hamsters was similar to that in castrates, indicating a lack of uterotropic steroid secretion. DISCUSSION The appearance and disappearance of HSD enzyme activity in thecal, granulosal, and luteal cells of the hamster ovary probably reflect changing ratios of gonadotropins and the resulting effects on ovarian development and steroid production. While interstitial /HSD did not exhibit obvious changes in activity, one cannot rule out variations in interstitial steroidogenic activity due to other enzymes not studied here. The present histochemical findings can be correlated with information on gonadotropins and ovarian functions in the golden Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18
7 All figures show the deep-blue formazan precipitate in areas of &-fi-hydroxysteroid dehydrogenase activity. A light neutral red counterstain was used. Yellow and orange filters were used for photography so as to increase contrast between formazan and the counterstain. Abbreviations: corpus luteum, CL; interstitium, I; granulosa, G; theca, T. FIG. I. Day I of the estrous cycle, about 1-1 hr after ovulation. A newly formed corpus luteum has some HSD activity. The theca of a growing follicle has activity about equal to that of the interstitial cells. The same condition is found on Day I of pregnancy. X6. FIG.. Day of the cycle. Strong activity is present in a corpus luteum, and there is very weak activity in the theca of a growing follicle. X6. FIG.. Day of the cycle The HSD in the corpus luteum has declined to the level of activity seen in the interstitium. X6. FIG.. Day of the cycle. A growing follicle has fhsd in the theca which has increased to the level of activity seen in the interstitium. X6. FIG. S. Day of the cycle during the late light period (proestrus). Enzyme activity has increased in the theca of a preovulatory follicle, and there is some activity in the granulosa for the first time during the estrous cycle. X6. FIG. 6. Corpora lutea on Day of the cycle at preovulatory estrus with very little enzyme activity remaining. X1. FIG. 7. Day at estrus. A portion of a preovulatory follicle has strong enzyme activity in the theca, and the granulosa contains IHSD of about equal intensity to the interstitium. X. 6S Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18
FIG. 8. Day of pregnancy. Differs from Day of the cycle by the presence of,hsd activity in the theca of the growing follicle at an intensity comparable to that of the interstitium. Corpora lutea have considerable enzyme activity. XS. FIG. 9. Day 8 of pregnancy, with a strong reaction in the corpus luteum. Moderate activity is present in the theca and granulosa of a growing follicle and in the interstitium. X7. FIG. 1. Day 1 of pregnancy, with the granulosa of growing follicles as active as the corpus luteum. x7. FIG. 11. Day 16 of pregnancy, shortly before parturition. The granulosa is still active, but the corpus luteum now has very little enzyme activity. X7. FIG. 1. Lactation, I day after delivery. The corpus luteum of pregnancy is negative. The granulosa of follicles from pregnancy also has little remaining enzyme activity. Patches of active interstitium are seen around these structures. X. FIG. 1. After 8 days of lactation, the ovary has no antral follicles, no corpora lutea, and HSD is found only in the interstitium. Xl8. 66 Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18
OVARIAN HSD IN THE HAMSTER 67 hamster. An ovulatory surge of LH occurs during the afternoon of proestrus (Orsini and Schwartz, 1966; Goldman and Porter, 197). FSH may also be involved in stimulating ovulation (Goldman and Mahesh, 1969). The increased IHSD activity in thecal cells and the appearance of this enzyme in granulosa cells would appear to be a follicular response to the release of these gonadotropins during proestrus (Day ). A luteolytic effect of preovulatory LH (Choudary and Greenwald, 1968) may accelerate the disappearance of luteal $HSD at estrus. However, luteal (HSD activity begins to decline on Day of the cycle. A stimulatory action of the proestrous LI-I surge on the interstitium was not manifested by an obvious change in the HSD reaction. An increased function of corpora lutea on Day of the cycle was indicated by a strong IHSD reaction in these structures. A moderate increase in plasma and luteal progesterone on this day was reported by Lukaszewska and Greenwald (197). Circulating progesterone is elevated again before ovulation on Day (Leavitt and Blaha, 1971; Lukaszewska and Greenwald, 197) when corpora lutea are negative for,hsd. Since follicles appear to be the most active at estrus we suggest they are an important source of preovulatory progesterone. Lukaszewska and Greenwald (197) unilaterally irradiated ovaries on Day to destroy follicles and still found an increase in ovarian progesterone on Day, suggesting interstitium as a source of preovulatory progesterone. If true, this would imply a different function of the interstitium at estrus than at other times of the cycle, but this could not be demonstrated by the histochemical procedures used in the present study. The gonadotropic complex which maintains pregnancy in the hamster appears to be FSH and prolactin, with small amounts of LH (Greenwald, 1967). Ovarian fhsd activity reflects these gonadotropins. Large antral follicles develop (FSH) and corpora lutea exhibit strong HSD to Day 1 (FSH and prolactin). The slight to moderate IIHSD activity in the granulosa of large follicles on Days -8 of pregnancy is accompanied by only a moderate activity in the theca. This differs from the response of follicles on Day of the cycle (FSH and LH surge) where intense thecal HSD activity accompanies a moderate granulosal activity. The increase of IIHSD activity in the granulosa during the latter part of pregnancy, as well as more luteal growth, could result from stimulation by placental gonadotropin (Lukaszewska and Greenwald, 1969). If the increased activity of granulosa is associated with estrogen secretion, as suggested by work done with other species (Ryan and Short, 196; Ryan, Petro, and Kaiser, 1968), this could inhibit pituitary FSH secretion, leading to the luteal decline seen on the last day of pregnancy before delivery. Corpora lutea and large follicles are absent during lactation. Only interstitium has ihsd activity, and this is apparently under the influence of LH (Greenwald, 196). While it could not be ascertained histochemically that the intensity of interstitial (HSD was greater during lactation, the total amount of interstitium was increased as shown histologically and by ovarian weight. The lack of uterine maintenance by the welldeveloped interstitium raises a question as to what its secretory activity may be, since lactation can continue in ovariectomized hamsters (Greenwald, 196). We thank Mrs. Joyce D. Collier for her able technical ACKNOWLEDGMENT assistance. REFERENCES CHOUDARY, J. B., AND GREENWALD, G. S. (1968). Comparison of the luteolytic action of LH and estrogen in the hamster. Endocrino!ogy 8, 19-16. FItoNK, A. H., AND FRAPS, R. M. (19). Induction of estrus in the ovariectomized golden hamster. Endocrino!ogy 7, 7-61. Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18
68 BLAHA AND LEAVITF GOLDMAN, B. D., AND MAHESH, V. B. (1969). A possible role of acute FSH release in ovulation in the hamster, as demonstrated by utilization of antibodies to LH and FSH. Endocrino!ogy 8, 6-. GOLDMAN, B. D., AND PORTER, J. C. (197). Serum LH levels in intact and castrated golden hamsters. Endocrinology 87, 676-679. GREENWALD, G. S. (196). Histologic transformation of the ovary of the lactating hamster. Endocrino!ogy 77, 61-6. GREENWALD, G. S. (1967). Luteotropic complex of the hamster. Endocrino!ogy 8, 118-1. LEAVITF, W. W., AND BLAHA, G. C. (197). Circulating progesterone levels in the golden hamster during the estrous cycle, pregnancy, and lactation. Biol. Reprod., -61. LUKASZEWSKA, J. H., AND GREENWALD, G. S. (1969). Comparison of luteal function in pseudopregnant and pregnant hamsters. J. Reprod. Fert., 18-187. LUKASZEWSKA, J. H., AND GREENWALD, G. S. (197). Progesterone levels in the cycling and pregnant hamster. Endocrinology 86, 1-9. ORINI, M. W., AND PSYCHOYOS, A. (196). Implantation of blastocysts transferred into progesterone treated virgin hamsters previously ovariectomized. J. Rep rod. Fert. 1, -1. ORINI, M. W., AND SCHWARTZ, N. B. (1966) Pituitary LH content during the estrous cycle in female hamsters: comparisons with male and acyclic females. Endocrinology 78, -. PRASAD, M. R. N., ORSm1I, M. W., AND MEYER, R. K. (196). Nidation in progesterone treated, estrogen deficient hamsters. Proc. Soc. Exp. Bio!. Med. 1, 8-1. PUPKIN, M., BRATF, H., WEISZ, J., LLOYD, C. W., AND BALOGH, K. (1966). Dehydrogenases in the rat ovary. I. A histochemical study of -- and a-hydroxysteroid dehydrogenases and enzymes of carbohydrate oxidation during the estrous cycle. Endocrinology 79, 16-7. RYAN, K. J., PETRO, Z. AND KAISER, J. (1968). Steroid formation by isolated and recombined ovarian granulosa and thecal cells..1. C/in. Endocrinol. 8, -8. RYAN, K. J., AND SHORT, R. V. (196). Formation of estradiol by granulosa and theca cells of the equine ovarian follicle. Endocrinology 76, 18-11. WATrENBERG, L. W. (198). Microscopic histochemical demonstration of steroid 9-ol dehydrogenase. J. Histochem. Cytochem. 6, -. Downloaded from https://academic.oup.com/biolreprod/article-abstract///6/7687 on July 18