Hypophysial Homogenate Research Articles

Steroidogenesis by ovaries and testes of the European catfish, the wels (Silurus glanis), in vitro

Testosterone, 3α,17-dihydroxy-5β-pregnen-20-one, 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and 5β-pregnane-3α,17,20β-triol were identified as the major metabolites of [(3)H] 17-hydroxyprogesterone in ovarian incubations of the European catfish Silurus glanis. 17,20βP and the reduced triol were present only in ovaries from fish primed with carp hypophysial homogenate (chh) while testosterone yields were significantly higher in controls than in treated fish. 11-Ketotestosterone, 11β-hydroxytestosterone and 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) were identified as the major metabolites of [(3)H]17-hydroxyprogesterone in in vitro incubations of testes of a spermiating catfish. There was no significant production of conjugates or other water soluble metabolites by either sex. The stimulation of plasma 17,20αP, 17,20βP and 11β-hydroxytestosterone by chh in primed but not control males suggests that the role of these steroids in spermiation should be further examined.

Sperm production and steroidogenesis in testes of the common carp, Cyprinus carpio L., at different stages of maturation

Testes from carp, Cyprinus carpio L., at five different maturational stages from immature through to spermiation and regression were incubated with or without addition of carp hypophysial homogenate (chh) for 8 or 20 h. Concentrations of steroids and spermatozoa were measured in the medium and the residual tissue examined histologically. There was an increase in the area of the germinal cysts containing spermatozoa, the percentage of the testis which they occupied and in the production of spermatozoa as the gonadosomatic index (GSI) increased, but this was unaffected either by incubation or by pretreatment with chh. The major steroid in plasma and in in vitro testicular cultures from all of the maturing fish captured in winter was 1 I‐ketotestosterone. The production rate of this steroid in virro was unaffected by GSI, while plasma levels tended to increase with GSI. 17.20β‐Dihydroxy‐4‐pregnen‐3‐one was detectable in significant amounts in only a few spermiating fish in summer, but was stimulated more in incubations with chh in maturing winter than in summer spermiating or post‐spawning fish. 17,20a‐Dihydroxy‐4‐pregnen‐3‐one was not detectable in incubations, but plasma concentrations tended to increase towards spermiation and were positively correlated with the size of the cyst. After spawning, fish had low plasma steroid levels and failed to respond in vitro to pituitary extract, indicating a testicular post‐spawning refractoriness.

Interactions between oocytes of different maturational stages in the carp Cyprinus carpio: Effects on maturational and steroidogenic activity in vitro

Ovarian fragments from carp which had received injections of saline (control) or carp hypophysial homogenate (primed) were incubated both alone and as cocultures of primed and control tissue in the presence of carp pituitary extract. The results showed that the cocultures were not simply the sums of separate incubations. Oocyte maturation was retarded in primed tissue but advanced in control tissue and concentrations of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) in the medium were significantly lower than that expected from separate incubations. The results indicate that local factors may act to synchronise oocyte maturation throughout the ovary to enable spawning of the maximum number of eggs.

The temporal sequence of changes in oocyte maturation and ovarian steroid hormone production during induced ovulation in the common carp, Cyprinus carpio

Oocytes taken from carp sacrificed 3–24 hr after injection with either carp hypophysial homogenate (chh) (primed) or saline (control) were incubated with or without chh for 24 hr at 20°. In oocytes from the primed fish the percentage of peripheral germinal vesicles at sacrifice progressively increased with time from injection. No such change occurred in control fish. After incubation with chh, tissue from primed fish showed a progressive increase in oocyte maturity with increasing time between priming and sacrifice, with germinal vesicle breakdown (GVBD) in 50% of the oocytes in incubations begun 24 hr after priming. In contrast, incubated tissue from control fish showed no variation with time between saline injection and sacrifice, and oocyte maturity did not exceed 50% with peripheral germinal vesicles. GVBD was not significant in these fish. 17-Hydroxyprogesterone, testosterone, and testosterone glucuronide production in vitro showed no major changes with the interval between injection and sacrifice, nor between control and primed fish. In vitro production of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), however, increased significantly between tissue taken from animals at 3 and at 6 hr after injection of chh, and thereafter remained unchanged. 17,20βP production preceded the increase in GVBD in primed fish but neither this hormone nor GVBD was found in incubations of control fish, supporting the suggestion that 17,20βP is the maturation-inducing hormone in carp. Steroid production was not detectable in the absence of chh in the incubation medium and oocyte maturation showed little advance on that at sacrifice. The results suggest that migration of the germinal vesicle and induction of 17,20βP potential by the priming dose are independent events and they are discussed in the context of the failure of some fish to ovulate despite apparently high initial oocyte maturity.

Gonadotropin-induced changes in steroid production by ovaries of the common careCyprinus carnio L. around the time of ovulation

Ovarian fragments from both primed (gonadotrophin treated) and unprimed female carp were incubated either with or without carp hypophysial homogenate and steroid hormone production measured. In incubations without hypophysial homogenate, production of all the steroids measured was either very low or nondetectable and there was no significant difference between tissue from primed and unprimed fish. In the presence of carp hypophysial homogenate a very significant increase in production of testosterone, 17-hydroxyprogesterone and testosterone glucuronide was observed, but there was no significant difference between primed and unprimed fish. 17,20β-Dihydroxy-4-pregnen-3-one (17,20βP) was not stimulated by carp hypophysial homogenatein vitro in ovaries from unprimed fish, but a very significant increase in production of this hormone was observed in tissue from fish which had received a priming dose of pituitary hormone. It is suggested that the priming dose of pituitary extract used in the normal hypophysation procedure to induce ovulation in teleosts initiates the potential for synthesis of 17,20βP in response to later gonadotrophin challenge, and that this initiation may be related to the migration of the germinal vesicle.

Autoradiographic localization of gonadotrophin receptors in ovaries of the common carp, Cyprinus carpio L

Binding sites for carp gonadotrophin have been located in carp ovaries using [ 125I]labeled gonadotrophin and autoradiography. The radioactive gonadotrophin was displaced from tissue by unlabeled gonadotrophin or carp hypophysial homogenate in a dose-dependent fashion. No binding of gonadotrophin was found in previtellogenic oocytes but binding appeared with the first indications of vitellogenesis. In the smaller vitellogenic oocytes binding was uniformly distributed in the follicular envelope, but in the largest oocytes binding was restricted to the interstitial tissue. In these more mature oocytes gonadotrophin was also found within the oocyte and appeared to migrate toward the nucleus. The relationship between binding location, steroidogenesis, and oocyte maturation is discussed. We found no evidence for specific binding of [ 125I]thyroxine under comparable conditions.

The effects of low dosages of steroid hormones and carp hypothysial homogenate on carp ( Cyprinus carpio) oocyte maturation in vitro

Using in vitro incubation of carp ovarian follicles, it was shown that low dosages of steroid hormones (0.1–0.5 μg/ml of medium) applied together with carp hypophysial homogenate (c.h.h.) at 100 μg/ml of medium exerted a much more potent effect on intrafollicular oocyte maturation than c.h.h. alone at 100 μg/ml or steroid hormones alone at 0.1–0.5 μg/ml. It is possible that the low dosages of steroid hormones or c.h.h. increased the sensitivity of the follicles to the final gonadotropin stimulation.

Effect of temperature, 17α-hydroxy-20β-dihydroprogesterone and prostaglandin F 2α on carp oocyte maturation and ovulation in vitro

Two series of experiments were carried out. In series I, studies were conducted on the effect of 17α-hydroxy-20β-dihydroprogesterone (17α20βP; 1 μg/ml of substrate) on the migration of germinal vesicle (GV) and its breakdown (BD) in the oocytes of carp ovarian fragments, kept at 5, 10, 15, 20, and 25°C. For investigations ovarian fragments were taken both from intact mature females and from those injected with carp hypophysial homogenate (chh) at a dose of 1 mg/kg. It was found that GV migration and GVBD occurred at 15–25°C. Based on the results obtained, it appears that GV migration proceeds optimally at 15°C, whereas GVBD requires higher temperatures (20–25°C), although at times it may also take place at 15°C. In series II, studies were conducted on the effects of exogenous prostaglandin F 2α (PGF 2α) on carp oocytes ovulation in vitro in the ovarian fragments kept at 5, 10, 15, 20, and 25°C for 24 and 48 hr. It was found that the temperatures in the range studied had no statistically significant effect on the ovulation process induced in vitro by exogenic PGF 2α added to the medium. The mechanisms of the final stage of carp oocytes maturation and ovulation are discussed.

Daily changes in the gonadotropin levels and response of carp oocytes to hypophysial homogenate.

Daily changes in carp gonadotropin levels in adult female carp and daily changes in carp oocyte sensitivity to carp hypophysial homogenate, in vitro and in vivo, were investigated. A total of three series of experiments were carried out. Gonadotropin levels were radioimmunologically determined. The results of series 1 and 2 experiments were subjected to statistical analysis with the use of cosinors circle and ellipse of errors. It has been found that in the mature female carp in the pre-spawning period with the light periods being long (L:D = 16:8) the apogee for gonadotropin occurs 10 hr after the onset of the light period. The sensitivity of the oocytes, in terms of the percentage of mature oocytes (after GVBD) following a 24-hr incubation of ovarian fragments with the hypophysial homogenate, reached the highest value at 1300, i.e. 9 hr after the onset of the light period. It was also found that the injections of carp hypophysial homogenate made at 0900 were much more efficient in inducing ovulation than those at 2100.

The effect of low doses of carp hypophysial homogenate and some steroid hormones on carp ( Cyprinus carpio L.) oocyte maturation in vitro

Studies on the in vitro incubation of carp oocytes have shown that low doses (10.0–50.0 μg/ml of medium) of carp hypophysial homogenate (c.h.h.) do not induce complete maturation. Steroid hormones at 1 μg/ml used alone stimulate only a percentage of oocytes to reach full maturation. Low doses of carp hypophysial homogenate (added to the culture at the beginning of incubation) and steroid hormones (added 6 h later) induce maturation in over 40% of oocytes. The highest percentage of mature oocytes was obtained in the cultures to which were added carp hypophysial homogenate at 30–40 μg/ml of medium and 17α-hydroxy-20β-dihydroprogesterone (17α20βP) (1 μg/ml of medium) 6h later. The results are discussed in connection with their application in artificial spawning of carp.

Changes in blood gonadotropin level in mature female carp following hypophysial homogenate injections

A hypophysial homogenate injection increased blood gonadotropin level in adult female carp and maintained it at that level for three days after injection. The blood gonadotropin level in adult female carp seems to be dependent on the physiological condition of the ovary expressed by the relation of old oocytes with a completed vitellogenesis process to the new ones, and the relation of the oocytes with the nucleus shifted to the periphery to those with a centrally placed nucleus.

Changes in the ovaries of adult carp

Three series of experiments were carried out. In the first, studies were conducted on the changes occurring in the ovaries of four groups of adult female carp during a yearly cycle (between two spawning periods). Gonadal samples in vivo were taken monthly from the same marked females from August 1975 to July 1976. A portion of the samples was subjected to histological examination and the rest used for counting and measuring oocytes. It was found that changes in the adult carp ovaries during a yearly cycle consist of a gradual development of a resorption process in the oocytes after a completed vitellogenesis, the store of oocytes being progressively supplemented by those in earlier stages of development. The changes studied are not temperature-dependent and their course is similar, irrespective of whether the females had or had not spawned during the spawning season preceding the experimental period. In the second series, studies were conducted on 26 marked, mature female carp, for 5 weeks, starting on the day before artificial spawning. Gonadal samples were taken in vitro, analysed histologically, a portion being cleared in turpentine oil while others were used for in vitro culture. It was found that, irrespective of whether the females had spawned or not, they possessed oocytes after a completed process of vitellogenesis. Their response to hypophysial homogenate added to the culture medium resulted in nucleus migration. In the third series, studies were conducted on the effect of clomiphene on the ovaries. It was found that clomiphene given to mature female carp during the spawning period caused a statistically significant increase, compared to the control, in the percentage of oocytes with the nucleus shifted peripherally. However, in contrast to hypophysial homogenate injections, it failed to induce ovulation.

Association of luteinizing hormone-releasing factor with granules separated from equine hypophysial stalk.

Axons of the external layer of the equine hypophysial stalk contained vesicles of 60–250 m/i in diameter. These vesicles were sedimented at 40,000 × g by differential centrifugation of the hypophysial stalk homogenate in a 0.44M sucrose solution. The sediment containing these vesicles had high luteinizing hormone-releasing factor (LRF) activity. After centrifuging a suspension of the sediment through a concentration gradient of sucrose, LRF activity was concentrated in a layer ranging from 1.2 to 1.6M in sucrose concentration. Activity of the neurohypophysial hormones was concentrated in the lower portion of the same layer. These results indicate that LRF, as well as the neurohypophysial hormones, is contained in the vesicles of the median eminence. (Endocrinology 86: 207, 1970)