Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.     

Treatment of rats with hCG induces inflammation-like changes in the testicular microcirculation

Adult rats were injected subcutaneously with 50 i.u. hCG and vascular permeability was compared to that in saline-treated control rats by two independent methods. At 4 h after hCG treatment the rats were injected intra-arterially (i.a.) with FITC-labelled macromolecular dextran (Mr 150,000) and the testicular microcirculation was studied in vivo by using a fluorescence microscope. Other rats were injected i.a. with a suspension of colloidal carbon and the location of leaking blood vessels was recorded in sections from the testes by light and electron microscopy. In hCG-treated animals leucocytes were found adhering to the endothelium in post-capillary venules and in these venular segments dextran was leaking into the interstitium. Carbon particles were deposited in the walls of post-capillary venules and leucocytes migrated through open interendothelial cell gaps in hCG-treated animals. In control animals leucocyte adhesion and migration were not observed, the injected dextran remained in the circulation and the blood vessels were not labelled by carbon. It is suggested that the hCG-induced increase in testicular interstitial fluid volume, like the tissue oedema in inflammation, is caused by a leucocyte-mediated increase in venular permeability.     

Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract

Previously, we described the presence of a factor obtained from a 105,000 X g supernatant of rat testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the present study, similarly prepared testicular extract was tested for its effects on in vitro hCG-stimulated testosterone production by isolated testis interstitial cells and for its effect on spontaneous ovulation in the rat. Incubation of interstitial cells with charcoal-treated extract significantly inhibited the steroidogenic response to hCG in a dose-related manner. This inhibition was also apparent after heating the extract for 10 min at 100 degrees C. Preincubation of the cells with charcoal-treated extract resulted in an inhibitory effect that was not readily reversed by subsequent addition of hCG, revealing an element of irreversibility in the mechanism of inhibition. A single i.p. injection of testicular extract given between 1430-1630 h of proestrus inhibited spontaneous ovulation in the rat. This effect was also observed after heating the extract for 10 min at 100 degrees C; in contrast, no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG after pretreatment with the testicular extract did not reverse the inhibitory effect on ovulation, indicating that this effect was probably not exerted at the hypothalamus-pituitary level. It is concluded that the aqueous testicular extract contains a factor able to antagonize the physiological events mediated by luteinizing hormone (LH)/hCG, and that this factor is consistent with the presence of an LH/hCG-binding inhibitory activity in rat testis.     

Human chorionic gonadotropin increases the concentration of macrophages in neonatal rat testis

The purpose of these studies was to determine whether treatment of newborn rats with exogenous FSH or hCG would alter the concentration or size of testicular macrophages. Animals were injected once daily with various doses of FSH, hCG, or vehicle for 8-10 days beginning the day following birth. After immunohistochemical labeling of the macrophages with a monoclonal antibody specific for rat macrophages, the concentration and size of macrophages were determined by use of a point-counting method. Body weight, testis weight, and serum levels of testosterone and FSH were also measured. It was found that hCG significantly increased the concentration of macrophages within the interstitium but did not affect the size of the cells. Both testicular weight and serum testosterone concentrations increased after hCG treatment. Although FSH increased the weight of the testis, neither the size nor concentration of macrophages was altered. These results raise the possibility that the number of macrophages within the interstitial compartment of the normally maturing rat testis is under the control of LH.     

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

Effects of PRL and FSH on LH binding and number of Leydig cells in hypophysectomized mice

The effects of PRL and FSH on testicular LH receptors were studied in hypophysectomized (hypox) mice. From the eleventh day after hypophysectomy, they were given 100 micrograms ovine (o) PRL and/or 2 micrograms oFSH in two injections per day for 10 days. Hypophysectomy reduced the weight of testis, epididymides and seminal vesicles, and the LH binding to the testis. Treatment with oPRL and/or oFSH in hypox mice resulted in an increase in the weight of testis and epididymides, and the LH binding per testis. There was no difference between the testicular LH binding in oFSH- and oPRL-treated mice. Histological examination showed that oPRL and/or oFSH treatment in hypox mice restored normal spermatogenesis. Administration of oFSH to hypox mice led to an increase in the number of typical Leydig cells, whereas oPRL was not effective. These results suggest that either PRL or FSH stimulates the LH binding to the testis, but that the action of PRL and FSH on the increase in testicular LH binding is different.     

Localized increases in ovarian vascular permeability and leucocyte accumulation after induced ovulation in rabbits.

Colloidal carbon was injected i.v. in mature virgin rabbits at different times after induction of ovulation by human chorionic gonadotrophin (hCG, 100 iu) or mating. Before induction of ovulation, slight carbon leakage was observed in the inner vascular ring of the theca interna of antral follicles, but blood vessels in the other ovarian compartments were unstained. Between 4 and 10.5 h after hCG-treatment or mating, vascular leakage was most marked in the blood vessels of the interstitial gland and in the theca interna of antral follicles. Just before ovulation, carbon particles were observed between granulosa cells and some carbon was seeping into the follicular fluid of preruptured follicles. Vascular leakage was also observed over the follicle dome before rupture as well as at the dorsomedial junction between the mesovarium and the ovary. The blood vessels stained with carbon were 7-70 microns diameter, representing capillaries and postcapillary venules. About 6 h after hCG injection, an increased number of polymorphonuclear leucocytes migrated from the vessels of these ovarian compartments into the surrounding interstitial tissue. The number of leucocytes seen in the follicular wall and ovarian medulla increased markedly towards ovulation. During early corpus luteum formation, the number of leucocytes decreased markedly. The localized vascular changes seen after mating and hCG stimulation were similar to an inflammatory reaction and could form the basis for the formation of peritoneal exudate after ovulation in rabbits and periovulatory ascitic accumulation seen in the peritoneal cavity of women during the menstrual cycle.     

Differential effects of follicle-stimulating and luteinizing hormones on testosterone production by mouse testes

In adult mice, direct intratesticular injection of ovine follicle-stimulating hormone (o-FSH-13; AFP 2846-C, from NIAMDD, less than 1% LH contamination) at 10, 100 or 1000 ng significantly elevated concentrations of testosterone (T) within the testis. These effects were rapid, with peak values attained by 15 min, and transient, with return to values comparable to that in the contralateral, saline-injected testis within 90 min. Intratesticular injection of FSH (1 microgram) significantly increased testicular T levels in 15- and 60-day old mice. This contrasted with the effects of intratesticular administration of human chorionic gonadotropin (hCG), which stimulated T production significantly at 30 days of age through adulthood. In adult mice, the equivalent LH to the possible contamination in the FSH preparation (1 ng) had no effect. Intratesticular injection of 10 ng LH produced comparable stimulation to that by 100 ng FSH (approximately 7-fold). Systemic pre-treatment with a charcoal-treated porcine follicular fluid (PFF) extract for 2 days reduced plasma FSH levels [86 +/- 17 (5) vs 700 +/- 8 (6); P less than 0.05], but had no effect on plasma LH. Twenty-four hours after the last treatment, the response to intratesticular injection of hCG (2.5 mIU), FSH (100 ng) or LH (10 ng) was also significantly attenuated in these mice. Intratesticular injection of PFF had no direct effect on testicular T levels. In vitro T production in the presence of hCG, LH or FSH were differentially affected by the concentrations of calcium (Ca2+) or magnesium (Mg2+) in the incubation media. The stimulatory effects of FSH were apparent at significantly lower levels of Ca2+ or Mg2+, than were those of LH or hCG. The results of these studies indicate that FSH is capable of stimulating testicular T production. Furthermore, the responsiveness to FSH is qualitatively different than that to LH/hCG in terms of the age pattern, as well as the dependence on Ca2+ or Mg2+. In addition, plasma FSH levels appear to influence testicular responsiveness to direct exogenous administration of gonadotropins. These studies indicate that FSH stimulation of T production can be differentiated from those of LH, and that these effects of FSH can be observed under physiological conditions.     

Regulation of infant and developing rat testicular gonadotropin and prolactin receptors and steroidogenesis by treatments with human chorionic gonadotropin, gonadotropin-releasing hormone analogs, bromocriptine, prolactin, and estrogen

Infant (5-day-old) male rats were treated with hormonal regimens to alter their exposure to gonadotropins, prolactin (Prl), and estrogen, and the response of testicular endocrine functions was measured. Human chorionic gonadotropin (hCG) or a potent gonadotropin-releasing hormone agonist analog (GnRH-A) resulted in a short-lived decrease of testicular receptors (R) for luteinizing hormone (LH), but no deleterious effects were found on testicular capacity to produce testosterone (T), which is a typical response of the adult testis. Only GnRH-A, through probable direct testicular action, induced a relative blockade of C21 steroid side-chain cleavage that was observed in vitro upon hCG stimulation. Human chorionic gonadotropin treatment, but not GnRH-A treatment, increased testicular Prl-R. GnRH antagonist analog (GnRH-Ant) treatment did not affect testicular LH-R, but decreased Prl-R and testicular T production. Decrease of serum Prl by bromocriptine had no effect on testicular LH-R or Prl-R, but slightly decreased T production in vitro. Ovine Prl increased binding sites for LH/hCG. The postnatal rats were insensitive to negative effects of diethylstilbestrol when monitored by testis weight, T, and LH-R. In conclusion, the responses to changes in the hormonal environment differed greatly between infant and adult testes. Mainly positive effects of elevated gonadotropin and Prl levels were seen on infant rat Leydig cell functions. Likewise, decreased tropic hormone levels, and exposure to estrogen, were ineffective in bringing about the inhibitory actions seen in the adult.     

Effects of photoperiod and hCG on the regulation of testicular LH/hCG receptors in Syrian hamsters (Mesocricetus auratus)

The regulation of testicular LH/hCG receptors was studied in Syrian (golden) hamsters with testicular atrophy induced by exposure to short photoperiod (5L:19D) and in gonadally active hamsters kept in a long photoperiod (14L:10D). By 24 h after injection of hCG, long-photoperiod hamsters showed a dose-related decrease in the number of testicular LH/hCG receptors. At 48 and 72 h, there was a recovery from this 'down-regulation'. The recovery was much faster than has been reported for the rat and mouse, and it resulted in elevation of testicular LH/hCG receptor concentrations above basal values. Hamsters with short photoperiod-induced testicular atrophy showed an increase in testicular LH/hCG receptors after injection of hCG, except for animals injected with a very high dose. The hCG-induced increase in testicular LH/hCG binding in these animals was associated with reappearance of testosterone responses to subsequent hCG stimulation. Response of testicular LH/hCG receptors to hCG in prepubertal hamsters resembled that measured in animals with short photoperiod-induced gonadal atrophy.     

Bisphenol A inhibits testicular functions and increases luteinizing hormone secretion in adult male rats

Effects of a xenobiotic estrogen, bisphenol A (BPA), on reproductive functions were investigated using adult male rats. BPA was dissolved into sesame oil and injected s.c. every day (1 mg/rat) for 14 days. Animals were killed by decapitation after the final administration of BPA, and the trunk blood, pituitary, and testes were collected. Plasma concentrations of prolactin were dramatically increased and pituitary contents of prolactin were slightly increased in the BPA group compared to the control group. Plasma concentrations of testosterone were decreased and plasma concentrations of LH were increased in BPA-treated rats compared to control rats. Testicular contents of inhibin were decreased in BPA-treated rats compared to control rats, although plasma concentrations of inhibin were not changed after administration of BPA. The testicular response to hCG for progesterone and testosterone release was decreased in BPA-treated rats. Administration of BPA did not change the pituitary response to luteinizing hormone-releasing hormone (LH-RH) in castrated male rats treated with testosterone. Male sexual behavior also was not changed as a result of BPA treatment. These results suggest that BPA directly inhibits testicular functions and the increased level of plasma LH is probably due to a reduction in the negative feedback regulation by testosterone. The testis is probably a more sensitive site for BPA action than the hypothalamus-pituitary axis.     

Further characterization of the direct inhibitory effect of LHRH agonists at the testicular level in the rat

To further clarify the relative importance of the pituitary and gonadal sites of LHRH action, intact and hypophysectomized adult male rats were treated with hCG for 7 days, in the presence or absence of simultaneous treatment with increasing doses of the LHRH agonist [D-Ser(TBU)6des-Gly-NH2(10)]LHRH ethylamide, Buserelin (0.025, 0.25, 2.5 or 25 micrograms/rat, twice daily). Daily treatment of intact adult rats with hCG (25 IU) markedly increased ventral prostate and seminal vesicle weight, while a dose-dependent inhibition of the effect was observed following combined administration of Buserelin. In hypophysectomized rats, treatment with hCG resulted in a partial restoration of ventral prostate and seminal vesicle weight, while combined treatment with a high dose of the LHRH agonist (25 micrograms, twice daily) partially (P less than 0.05) inhibited the stimulatory effect of hCG. LH/hCG receptors were almost completely inhibited after hCG injection alone and a further decrease was observed in the presence of simultaneous LHRH agonist treatment. The hCG-induced stimulation of GH/PRL receptors was counteracted by Buserelin treatment in hypophysectomized animals. The present data demonstrate that although LHRH-induced LH release has been shown to play a major role in the loss of testicular functions induced by low doses of LHRH agonists in the rat, a direct inhibitory action of LHRH agonists can be exerted at the testicular level at high doses of the peptide.     

Differential effects of follicle-stimulating hormone and luteinizing hormone on Leydig cell function and restoration of spermatogenesis in hypophysectomized and photoinhibited Djungarian hamsters (Phodopus sungorus)

Effects of pure human follicle-stimulating hormone (hFSH) and ovine luteinizing hormone (oLH) on testicular function were investigated in long-term hypophysectomized or photoinhibited Djungarian hamsters. hFSH (5 IU) or oLH (5 micrograms) or a combination of FSH and LH (5 IU and 5 micrograms, respectively) were injected s.c. twice daily for 7 days to hypophysectomized and photoinhibited hamsters. Other photoinhibited hamsters were treated for 14 and 21 days with FSH and LH (3 IU and 3 micrograms, respectively) in a similar way. LH alone had little, if any, effect on testicular weights; FSH, when injected alone or in combination with LH (FSH/LH), caused a significant increase in testes weights at each time point. On the other hand, LH or FSH/LH, but not FSH alone, caused a significant increase in the accessory organ weights. FSH had no effect on intratesticular testosterone (T) or on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity but enhanced the in vitro response of interstitial cells to hCG. LH and FSH/LH had pronounced effects on intratesticular T, 3 beta-HSD activity, and in vitro response of interstitial cells to human chorionic gonadotropin. Treatment with FSH or FSH/LH caused regrowth of the testis and restoration of tubular lumen and tubular diameter and restored complete spermatogenesis. However, LH had little effect on spermatogenesis in spite of increased intratesticular and peripheral T levels. These results indicate that although LH can cause a full redifferentiation of Leydig cells in photoinhibited hamsters, it has only minor effects on tubular function. On the other hand, FSH alone induces full restoration of tubular function in these animals and has no direct effect on Leydig cell steroidogenesis, but may enhance the Leydig cell responsiveness to LH.     

Impaired gonadotrophin uptake in vivo by the cryptorchid rat testis

The specific testicular uptake in vivo of 125I-labelled hCG was compared in control adult rats and adult rats made bilaterally cryptorchid 5 weeks previously. Although a similar temporal pattern of uptake was observed in both groups, uptake of hCG by cryptorchid testes was reduced at all times after injection by up to 70%. The possible causes of this impairment were investigated. It could not be accounted for by differences in the rate of absorption or clearance of 125I-labelled hCG in the two groups. Therefore, because hCG-induced increase in the permeability of testicular capillaries is a crucial factor in determining hCG uptake by the testis, this change was compared in control and cryptorchid testes. Although hCG induced a characteristic increase in testicular capillary wall permeability in both groups, this change was temporally delayed in cryptorchid testes, and occurred after hCG values in the blood had fallen. Even when hCG had crossed the capillary wall into testicular interstitial fluid, its uptake into the testicular tissue was significantly lower in cryptorchid than in control testes. These changes probably account for the impairment of gonadotrophin uptake by the cryptorchid testis and have important implications with respect to the aetiology of Leydig cell changes in cryptorchidism.     

Testicular blood flow and microcirculation in rats after treatment with ethane dimethyl sulfonate

The effects of ethane dimethyl sulfonate (EDS) on total testicular blood flow, microcirculation, and the testicular interstitial fluid volume (IFV) in rats were studied. In agreement with previous studies, treatment of control rats with human chorionic gonadotropin (hCG) induced an increase in IFV and total testicular blood flow as measured with radioactive microspheres. These effects of hCG were completely abolished in rats pretreated with EDS; in EDS-treated rats not receiving any hCG, there were decreases in IFV when compared with untreated control rats. Furthermore, the pulsatile pattern of testicular microcirculation registered with laser-Doppler flowmetry was abolished after EDS treatment, and this effect was not influenced by hCG treatment. The hCG-induced increase in IFV is associated with an increased accumulation of polymorphonuclear leukocytes locally in the testis, but this accumulation of leukocytes was not observed in rats pretreated with EDS. It was concluded from the present study that hCG-induced changes in total testicular blood flow and testicular microcirculation require functionally intact Leydig cells.     

Kinetic and autoradiographic studies on binding of hCG to the testicular LH receptors of neonatal rats

The properties of hCG binding to LH receptors of the neonatal (5-day-old) rat testis were analysed and compared with those of the adult testis. The equilibrium association constants (Ka) of hCG-binding were similar at both ages, 2-4 X 10(10) M-1. In contrast, kinetic binding studies revealed that the association and dissociation rate constants of hCG binding were more rapid in the neonatal testis. Likewise, it was observed that the progression from loose (easily dissociable) to tight (non-dissociable) binding was less complete in the young than in the adult testis. Autoradiography of 125I-labelled hCG binding to interstitial cell suspensions at the two ages showed that the gonadotrophin binding per Leydig cell was about 50% lower in the neonatal testis. Conversely, since the surface area of adult Leydig cells was about 4-fold larger, the receptor density appeared to be higher in the neonatal Leydig cells. The rapid recovery of LH receptors after hCG stimulation, typical of the neonatal cells, was due to rapid replenishment of binding in the cells initially occupied by the injected hormone, rather than to an hCG-induced increase of Leydig cell number. Finally, in-vivo experiments with cycloheximide revealed that the rapid recovery of LH receptors was dependent on protein synthesis. These differences in the kinetics of neonatal testicular LH receptor turnover may be involved in the unique functional features of the fetal-neonatal growth phase of rat testicular Leydig cells.     

Effects of different numbers of ectopic pituitary transplants on regulation of testicular LH/hCG and prolactin receptors in the hamster (Mesocricetus auratus)

Adult male hamsters were given transplants of 1/2, 1, 2, 3 or 4 pituitaries under the kidney capsule and were killed 4 weeks later. Pituitary transplants produced a significant, dose-related increase in plasma prolactin levels, no changes in plasma LH and an increase in plasma FSH. Concentration of LH/hCG receptors in the testes was significantly increased in animals with 2 or 3 transplants and concentration of testicular prolactin receptors was significantly increased in those given 2 transplants. The apparent stimulatory effects of 1/2, 1 or 4 transplants on testicular LH/hCG and prolactin binding were not statistically significant. Some of the animals were injected with 0.3 i.u. hCG/g body weight 24 h before being killed. This produced a significant reduction in the levels of prolactin receptors and an apparent reduction in the levels of LH/hCG receptors in the testes. Elevation of plasma testosterone concentrations in response to hCG was significantly greater in animals given 3 or 4 pituitary transplants than in the remaining groups. These results provide further evidence that prolactin increases the number of LH/hCG and prolactin receptors in the hamster testis and suggest that changing the number of ectopic pituitary transplants may result in biphasic effects on the testis, with 2 or 3 transplants being maximally stimulatory.