by isolated rabbit ovarian follicles

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1 Effects of pregnant mare's serum gonadotrophin administered in vivo on steroid accumulation by isolated rabbit ovarian follicles E. V. YoungLai Department Obstetrics and Gynecology, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 3Z5 Abstract. Various doses (5, 50 and 100 IU) of pregnant mare's serum gonadotrophin (PMS) were administered to sexually mature female rabbits and steroids measured in follicular fluid, ovarian pieces and in incubation media of isolated follicles treated with ovine luteinizing hormone (LH). Follicular fluid progesterone, oestradiol and androgen were increased after PMS injection. Ovarian progesterone content was increased by all doses of PMS, and androgen and oestradiol content only by 50 and 100 IU PMS. Medium sized follicles from rabbits treated with 50 and 100 IU PMS accumulated more progesterone than all follicles from rabbits treated with saline or 5 IU PMS. Androgen accumulation by small follicles from 50 and 100 IU dose rabbits was higher than that by follicles from other treatment groups. Oestradiol accumulation mimicked androgen accumulation. The addition of LH to the medium stimulated the accumulation of progesterone and androgen by all follicles. LH caused an increase in oestradiol accumulation by medium follicles from saline and low dose PMS treated animals. No effect of LH on oestrogen accumulation was seen with large or medium follicles from animals treated with 100 IU PMS or 50 IU PMS for 2 or 3 days. These results suggest that priming of small follicles with PMS can increase their ability to produce androgen which is probably aromatized to oestrogen. Pregnant mare's serum gonadotrophin (PMS) which contains both follicle stimulating hormone (FSH) and luteinizing hormone (LH) activities, has been widely used to stimulate follicular growth and development. In the immature female rat PMS can cause acute and chronic changes in ovarian ste roidogenesis and depending on the dose of PMS used ovulations may occur (Sashida & Johnson 1976; Parker et al. 1976; Bauminger et al. 1977). In these studies PMS in doses ranging from 5 to 20 IU per rat have been used. On the other hand the use of PMS in the rabbit to study steroidogenesis has been limited. The isolated rabbit ovarian follicle has been shown by a number of investigators to be a very active steroidogenic system producing progestins, androgens and oestrogens (Bahr et al. 1980; Mills 1975; Nicosia et al. 1975; YoungLai 1974). In these studies normal sexually mature rabbits of the New Zealand White strain have been used without any further treatment. In other stud ies it was felt that the prior administration of 50 IU of MSP to rabbits 3 5 months old would ensure that all animals were in a uniform endocrine status (Morris & Gorski 1973). The present study was therefore undertaken to determine the effects of various doses of PMS on steroid content, follicular fluid steroids and the response of isolated follicles tolh. Materials and Methods Sexually mature New Zealand White rabbits greater than 5 months old and weighing approximately 4 kg were used. They were housed individually in cages with food and water available ad libitum. PMS was obtained from Sigma and diluted in normal saline before use. Animals were injected sc with PMS and then killed 24 h after the last injection. These animals were then designated as PMS 5/4 for three injections of 5 IU PMS 24 h apart and killed the fourth day; PMS 50/2 for one injection of

2 50 IU PMS and killed the next day; PMS 50/3 for two injections of 50 IU 24 h apart and killed the third day; PMS 50/4 as PMS 5/4 but with 50 IU PMS and PMS 100/2 one injection of 100 IU PMS. Controls were untreated mature rabbits. Ovaries and uteri were removed and weighed. A piece of ovary from each rabbit was placed in Davidson's fixative for subsequent histological examina tion using haematoxylin and eosin staining. Follicles were then dissected free from the remaining ovary and pooled according to size: < 1 mm diameter small (S) follicles; 1 2 mm medium (M) follicles and > 2 mm large (L) follicles. Haemorrhagic follicles were not used. The fol licles were incubated singly or in groups of 2 6 for 1 h in Krebs Ringer bicarbonate buffer containing 200 mg/100 ml glucose and 0.1% bovine serum albumin (BSA). Media were removed and follicles incubated for a further 3 h in medium with or without 1 µg/ml LH (NIH-oLH- S17). The media were then removed and stored for later analysis. The follicles were washed 3 x in buffer without BSA and homogenized with a Polytron homogenizer for protein determination. Large follicles contained about 200 µg protein/follicle; medium about 45 µg and small 21 pg. Pieces of ovarian tissue were also frozen for subsequent steroid analyses. They were either pooled or analysed separately. Tissue was thawed, homogenized in buffer without BSA and various dilutions analysed for steroids. For some ovaries follicular fluid was aspirated with a 25 gauge needle and 1 ml syringe, from all follicles 1 mm diameter. The fluid was diluted with radioimmunoassay buffer and different dilutions analysed for progesterone, androgen and oestradiol. Results are expressed as total steroid/follicle. Radioimmunoassays Steroid assays were carried out on dilutions of incubation medium using antisera prepared in our laboratories. The progesterone antiserum was raised against progesterone- than 0.1% cross- llct-hemisuccinyl-bsa and had less reactivity with 20a-dihydroprogesterone, testosterone, dihydrotestosterone, androstenedione, oestrone and oestradiol. Only corticosterone cross-reacted 1.2%. The limit of sensitivity of the assay was 10 pg. Testosterone was measured using an antiserum prepared against testosterone-3-oxime-bsa (Armstrong et al. 1978). It crossreacted significantly with dihydrotestosterone (35%). Results are therefore expressed as androgens. The limit of sensitivity of the assay was 25 pg. Oestradiol was measured using an antiserum prepared against oestradiol-6-carboxymethyloxime-bsa (Wielgosz et al. 1980). Its cross-reaction with oestrone, oestriol, progesterone, testosterone, and androstenedione was less than 0.1%. The limit of sensitivity of the assay was 10 pg. All steroid assays had an inter-assay coefficient of variation of less than 15%. Statistical analysis values between PMS-treated groups were determined by analyses of variance followed by Duncan multiple range test using an HP-97 calculator. A value of 0.05 was considered significant. Paired Student's i-test was used to compare results of LH treatment with controls. Where variance was not homogenous, values were con verted to natural logarithms before statistical analyses. All differences noted were statistically significant unless otherwise noted. Results The weights of the rabbits used and their repro ductive organs are shown in Table 1 with the Table I. Weight of rabbits and reproductive organs. Group Treatment Body weight (kg) Uterine weight (g) Ovarian weight (mg) Control PMS 5/4 PMS 50/2 PMS 50/3 PMS 50/4 PMS 100/ ± 0.25 (n 6) 4.04 ±0.11 (n 4) 4.14 ± 0.09 (n 8) 4.05 ± 0.13 (n 8) 3.99 ± 0.09 (n 8) 4.19 ± 0.09 (n 11) 6.59 ± ± ± ± ± ± ± ± ± ± ±21 No significant difference in uterine weights between treatment groups. Ovarian weights of groups 4 6 though not significant from each other, were heavier than those of groups 1 3 (Duncan's multiple range test).

3 Table 2. Follicular fluid steroids of PMS treated rabbits. Group Steroid content (pg/follicle) Treatment Progesterone Androgen Oestradiol Control PMS 5/4 PMS 50/2 PMS 50/3 PMS 50/4 PMS 100/2 245 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 28 Follicular fluid was aspirated from all follicles > 1 mm diameter, and mixed with 200 µ radioimmunoassay buffer. Different aliquots were taken for assay follicles per group. Progesterone and androgen were consistently detected in all follicular fluid examined and were elevated after all PMS treatments. Follicular fluid oestradiol was significantly increased in groups 2 5 inclusive. number in each group listed. There was no signifi cant difference in body and uterine weights. The ovarian weights of rabbits in PMS 50/3, 50/4 and 100/2 were significantly different from those of other groups. All doses of PMS caused an increase in ovarian content of progesterone from 2.4 ng/mg protein to about 35 ng. Androgen content increased from 1.5 to 4-7 ng with 50 and 100 IU PMS. Oestradiol content was increased from 0.1 to 0.4 ng with PMS 50/3, 50/4 and 100/2. Follicular fluid was expressed from follicles 200 PROGEStERONE ACCUMUL (ng/mg Protein) S M PMS 50/2 PMS 50/3 i PMS 50A PMS IOO/2 Fig. L Progesterone accumulation in the medium from isolated follicles during 3 h incubation in the presence and absence of 1 µg LH/ml. Open bars represent values in the absence of LH, dark bars the presence of LH. S small follicles follicles >2 mm diameter. See text for <1 mm diameter. M medium follicles 1 2 mm diameter. L large description of treatment groups. Mean ± SEM for 4 11 incubations per group. In the absence of LH, the endogenous accumulation of progesterone by small follicles in PMS 50/3, 50/4 and 100/2 was higher than that by small follicles from controls, PMS 5/4 or 50/2 (Duncan multiple range test). All LH treatment groups were significantly different from incubations without LH (t-test).

4 > 1 mm diameter and diluted with radioimmuno were taken for assay buffer to 200 µ. Aliquots steroid analysis. As shown in Table 2 all doses of PMS caused increases in follicular fluid progester one and androgen. By contrast, the oestradiol content of follicular fluid was increased in all PMS treated follicles except those from PMS 100/2. Progesterone accumulation in the medium dur ing incubation of isolated follicles is shown in Fig. 1. Large follicles > 1 mm were only present in groups PMS 50/3, 50/4 and 100/2. The endogen ous accumulation of progesterone by S follicles was higher in these 3 groups than in controls or PMS 5/4 or 50/2 where levels were at the sensitivity of the assay. LH caused a greater increase in proge sterone accumulation by S and M follicles of con trols, PMS 5/4 and PMS 50/2 compared to that from PMS 50/3, 50/4 and 100/2. The large follicles from PMS 100/2 accumulated much more proge sterone than follicles from all other groups in the absence and presence of LH. Androgen accumulation by all follicles from PMS 50/3 and 50/4 was greater than controls and PMS 5/4 (Fig. 2). Marked variation was seen in the follicles from PMS 100/2. In this group small than M and L follicles produced more androgen follicles. The greatest response to LH was seen by all follicles from controls and PMS 5/4 where increases ranged from fold. All follicles from the 3 PMS 50 groups responded to LH with a 2 17-fold increase in androgen accumulation. In the PMS 100/2 group the M and L follicles were stimulated to release androgen about 2-fold com pared to 8-fold by S follicles. The accumulation of oestradiol by follicles is shown in Fig. 3. PMS at doses of 50 and 100 IU increased the ability of small follicles to accumulate oestradiol. LH had no effect on small follicles from controls, PMS 5/4, medium follicles from PMS 50/3, 50/4, 100/2 and all large follicles. LH caused a significant increase in oestrogen accumulation by medium follicles from controls, PMS 5/4, PMS 50/2 and small follicles from PMS 50/4 animals. Discussion The results of this study demonstrate marked alterations in the quality of steroids released by follicles isolated 24 h after PMS adminstration and incubated in vitro. Moreover, there was a graded effect on follicular size depending on dose sche dule. Any conclusions based on the quantitative analysis of steroids produced after such treatment must therefore be interpreted with caution. The most consistent effect of PMS seemed to be an increase in the progesterone content of the ovaries which was elevated at all doses of PMS. This may represent increased progestin synthesis by interstitial tissue which was well developed in ova rian sections examined histologically. PMS at 5 IU ANDROGEN ACCUMULATION (ng/mg Protein) I UUiiJ S M S M S M S Control PMS 5/4 PMS 50/2 PMS 50/3 PMS 50/4 PMS 100/2 Fig. 2. Androgen accumulation in the medium from isolated follicles during 3 h incubation in the presence and absence of 1 µg LH/ml. Symbols are identical to those of Fig. 1. All follicles from PMS 50/3 and 50/4 accumulated more androgen than controls or PMS 5/4 (multiple range test). LH caused a significant increase in accumulation in all except medium and large follicles in PMS 100/2 (paired 'f test).

5 ESTRADIOt ACCUMULATION (ng/mg Protein) S M Control S M PMS 5/4 PMS 50/2 PMS SO/3 PMS 50/4 PMS IOO/2 Fig. 3. Oestradiol accumulation in the medium from isolated follicles during 3 h incubation in the presence and absence of 1 µg LH/ml. Symbols are identical to those of Fig. 1. Fifty and 100 IU PMS caused small follicles to release more oestradiol than other groups where oestradiol was not detectable. LH caused a significant increase in oestrogen accumulation by medium follicles from controls, PMS 5/4, PMS 50/2 and small follicles from PMS 50/4 (r-test). per rabbit did not provide sufficient stimulus to alter the ovarian content of androgen and oestra diol. Higher doses were required to elicit an effect. The contributions of the interstitial cells and follic les to the changes in steroid content could not be assessed. However, it known that the follicles pro vide the major source of testosterone and oestra diol whereas progestins may also be derived from interstitial tissue (YoungLai 1974; Mills 1975; Hilliard & Eaton 1971; Hilliard et al. 1969). The increase in ovarian steroid content can therefore be attributable to a general stimulation by PMS. Levels of follicular fluid steroids in the control rabbits were similar to those reported by a number of authors (Nicosia et al. 1975; YoungLai 1972; Patwardhan & Lanthier 1976; Bahr 1978; LeFevre & Caillol 1978). The effects of PMS on follicular fluid steroid content have not hitherto been re ported. It has previously been found that an ovula tory dose of LH or mating will cause a short-term increase in follicular fluid steroids followed by a depression (YoungLai 1972; Patwardhan & Lan thier 1976; Bahr 1978). In the present study none of the doses of PMS induced ovulations. However, in the groups PMS 50/3, 50/4 and 100/2 there were several follicles greater than 2 mm diameter. The greatest increase in progesterone content was found in the PMS 100/2 group. PMS 50 seemed to be the optimum for increasing follicular fluid oestradiol levels. If oestradiol content can be corre lated with follicular maturation as it does in human (McNatty & Baird 1978) then the results of this study would suggest that 50 IU PMS may be opti mal for stimulating follicular maturation. It should be noted that while mating or an ovulatory dose of LH cause a 2 5-fold increase in follicular fluid oestradiol within 3 h (YoungLai 1972; Patwardhan & Lanthier 1976; Bahr 1978) 50 IU PMS caused a fold increase over a longer time period. It is possible that some of the large follicles may not be ovulatory. Follicular fluid androgen was also higher than that reported on stimulation with LH (Bahr 1978). In spite of the LH content of PMS ovulations were not elicited with 50 or 100 IU. The in vitro response of follicles to stimulation with LH was obviously markedly influenced by the prior treatment of varying doses of PMS (Figs. 1 3). With regard to progesterone accumulation

6 small follicles from PMS 50/3, 50/4 and 100/2 were more active than those from saline, PMS 5/4 and was observed in PMS 50/2. A graded response medium follicles. All large follicles accumulated substantial amounts of progesterone. Upon stimu lation with LH the most pronounced effects were observed in incubations with small follicles from saline treated, PMS 5/4, 50/2 and 100/2; medium follicles from control and large follicles from PMS 100/2. The high accumulation of progesterone by medium and large follicles from PMS 100/2 ani mals parallel the follicular fluid content (Table 2) and suggest that PMS can stimulate granulosa cell progesterone production. Whether PMS caused a shift in steroidogenesis from oestradiol to proge sterone production as seen in the phénobarbital treated hamster (Terranova 1981), could not be de termined. However, the follicles from PMS 100/2 were also accumulating oestradiol (Fig. 3) which was not greatly stimulated by LH. Androgen accumulation in the medium from all follicles was consistently low in all treatment groups. However, there were marked differences in the response of follicles to stimulation with LH (Fig. 2). All follicles from controls and PMS 5/4 had a greater than 50-fold increase in androgen accu mulation compared to an 8-fold increase in the other treatment groups. The other major differ ence was the loss of responsiveness of the medium and large follicles of the PMS 100/2 group to LH stimulation. This lack of responsiveness is similar to the effect seen by follicles isolated from rabbits mated 48 h previously (YoungLai 1977) and may indicate a desensitization phenomenon (Hunzicker-Dunn et al. 1979). But the obvious progester one response to LH by the same follicles (Fig. 1) argues against this process. The lack of stimulation of androgen and oestrogen in this group suggests a lesion in the steroidogenic pathway, perhaps at the Similar results C21-side chain cleavage enzyme. have been found in the rat follicle within 4 6 h of LH stimulation (Lieberman et al. 1975; Hillensjö et al. 1977). Oestradiol accumulation by all follicles was consi stently enhanced by prior treatment with 50 and 100 IU PMS. Since oestradiol production can serve as a good index of follicular maturation (McNatty & Baird 1978) these data suggest that PMS can increase the pool of maturing follicles in older rabbits. This is consistent with the observations of Kennelly & Foote (1965) that FSH administered to rabbits greater than 16 weeks old, increases the ovulation rate. In the rat, PMS can increase the for mation of LH receptors in small follicles (Richards et al. 1976). If this also occurs in the rabbit then the stimulation of oestradiol accumulation by LH is not surprising especially since it has been demonstrated using rigorous radiochemical techniques that LH can increase rabbit follicular oestrogen synthesis from acetate (Mills et al. 1971). The lack of further stimulation by LH with medium and large follicles from PMS 50/3, 50/4 and 100/2 may represent advanced maturation and occupation of receptor sites. These data suggest that the schedule of PMS administration can influence subsequent steroid release by isolated ovarian follicles, stimulate folli cular increase in size and increase ovarian and follicular content of steroids. References Armstrong R A, Gauldie J & YoungLai E V (1978): Effects of active immunization of female rabbits against testosterone. J Endocrinol 79: Bahr J M (1978): Simultaneous measurement of steroids in follicular fluid and ovarian venous blood in the rabbit. Biol Reprod 18: Bahr J, Gardner R, Schenck 8c Shahabi (1980): Follicular steroidogenesis: effect of reproductive con dition. Biol Reprod 22: BaumingerS, Eckstein & Lindner H R (1977): Changes in steroid concentration in the ovaries of immature rats treated with pregnant mare serum gonadotropin and human chorionic gonadotropin. J Endocrinol 77: Hillensjö Hamberger L & Ahren (1977): Effect of androgens on the biosynthesis of estradiol-17ß by isolated periovulatory rat follicles. Moll Cell Endo crinol 9: Hilliard J, Spies H G & Sawyer C H (1969): Hormonal factors regulating ovarian cholesterol mobilization and progestin secretion in intact and hypophysectomized rabbits. In: McKerns W (ed). The Gonads, pp Elsevier, Amsterdam. Hilliard J & Eaton L W Jr (1971): Estradiol-17ß, proge sterone and 20ct-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. II. From mating through im plantation. Endocrinology 89: Hunzicker-Dunn M, Jungman R A 8c Birnbaumer L (1979): Hormone action in ovarian follicles: adenylyl cylcase and protein kinase enzyme systems. In: Midgley A R Jr & Sadler W A (eds). Ovarian Follicular Development and Function, Raven Press, New York.

7 Kenellv J J Se Foote R H (1965): Superovulatory response of pre- and post-pubertal rabbits to commercially avail able gonadotrophins. J Reprod Fértil 9: LeFevre B Se Caillol M (1978): Relationship of estrous behaviour with follicular growth and sex steroid con centration in the follicular fluid in the domestic rabbit. Ann Biol Anim Biochim Biophys 18: Lieberman E, Barnea A, Bauminger S, Tsafriri A, Collins W P Se Lindner H R (1975): LH effect on pattern of steroidogenesis in cultured Graffian follicles of the rat: dependence on macromolecular synthesis. Endocrinology 96: McNatty & Baird D (1978): Relationship between follicle-stimulating hormone, androstenedione and oestradiol in human follicular fluid. J Endocrinol 76: Mills M (1975): Effect of luteinizing hormone and cyclic 3',5'-monophosphate on steroidogenesis in the ovarian follicle of the rabbit. Endocrinology 96: Mills M, Davies J A & Savard (1971): Stimulation of estrogen synthesis in rabbit follicles by luteinizing hormone. Endocrinology 88: Morris P W Se Gorski (1973): Control of steroidogenesis in preovulatory cells. Luteinizing hormone stimulation of [14C] acetate incorporation into cells. J Biol Chem 248: Nicosia S V, Eangelista L Se Batta S (1975): Rabbit ovarian folllicles. I. Isolation technique and characteri zation at different stages of development. Biol Reprod 13: Parker C R, Costoff A, Muldoon T G & Mahesh V C (1976): Actions of pregnant mare serum gonadotropin in the immature female rat: correlative changes in blood steroids, gonadotropins, and cytoplasmic steroid receptors of the anterior pituitary and hypothalamus. Endocrinology 98: Patwardhan V V & Lanthier A (1976): Effect of an ovulating dose of luteinizing hormone on the concen tration of oestrone, oestradiol and progesterone in the rabbit ovarian follicle. Acta Endocrinol (Copenh) 82: Richards J S, Ireland J J, Rao M C, Bernath G A, Midgley A RJr & Reichert L E R Jr (1976): Ovarian follicular development on the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteiniz ing hormone. Endocrinology 99: Shashida & Johnson D C (1976): The response of the immature rat ovary to gonadotrophins: acute changes in the cyclic AMP, progesterone, testosterone, androstenedione and oestradiol after treatment with PMS or FSH + LH. Acta Endocrinol (Copenh) 82: Terranova P F (1981): Steroidogenesis in experimentally induced atretic follicles of the hamster: a shift from estradiol to progesterone synthesis. Endocrinology 108: Wielgosz G, Low M J & YoungLai E V (1980): Effects of immunizing rabbits against oestradiol-6-oxime-bsa on ovarian follicular development. Acta Endocrinol (Copenh) 94: on follicular YoungLai E V (1972): Effects of mating fluid steroids in the rabbit. J Reprod Fértil 30: YoungLai E V (1974): Steroid production by the isolated rabbit ovarian follicle. I. Effects of ovine LH. J Reprod Fértil 40: YoungLai E V (1977): Steroid production by isolated rabbit ovarian follicles: effects of luteinizing hormone from mating to implantation. J Endocrinol 73: Received on May 30th, 1984.