Leydig cell clusters in adult testes enlarge considerably under treatment with excess LH or hCG. However, it has been uncertain in the past whether this expansion involves an increase in the number of Leydig cells or merely an enlargement of the individual cells. We have used morphometric methods to investigate this question in the testes of rats treated chronically with hCG. Adult male Sprague-Dawley rats were injected s.c. daily with 100 IU hCG for up to 5 weeks. Testes were fixed by perfusion with Bouin's fixative, embedded in paraffin, sectioned longitudinally and stained for light microscopy with Mallory azan. To avoid counting macrophages, which are also numerous in the interstitial tissue, the animals were injected with trypan blue before sacrifice. The number of Leydig cells and their volume was determined by morphometric methods (Weibel and Bolender, 1973). Appropriate normal controls were included. The volume of Leydig cell clusters increased by a factor of 4.7 during the 5 weeks of hCG treatment. The number of Leydig cells (initially averaging 18.6 x 10 6 /cm 3 testis) increased to 3 times the control value by 5 weeks of treatment (P<0.001), while the average volume of individual Leydig cells (initially â¼2200 Âµm 3 ) enlarged only 1.6 times. We conclude that chronic treatment with an excess of hCG increases the number of Leydig cells in the testes of adult rats and that this hyperplasia is more important than cell hypertrophy in the expansion of Leydig cell clusters.
An estrogen-induced protein has been isolated by a simple method from plasma of rainbow trout ( Salmo gairdneri ) and an antibody against it was raised in a rabbit. Three fractions were isolated from trout ovaries using the method for isolation of lipovitellin and phosvitin of Wallace et al. (1966). Two factors crossreacted with the antibody and could be displaced by serum from vitellogenic trout only, suggesting that the isolated serum fraction was trout vitellogenin. Biochemical analysis established similarities between vitellogenin, lipovitellin and phosvitin from trout and other oviparous species. A radioimmunoassay for vitellogenin has been developed for rainbow trout using vitellogenin as standard and gonad lipovitellin or Î²' component as radioligand. In this system little cross reactivity was found with vitellogenin rich sera from closely related Salmo salar .
During the epididymal transit of spermatozoa, their membranes become coated with glycoproteins, which may play an important role in fertilization. One of these glycoproteins has been purified from rat epididymides. It has a molecular weight of 37,500 daltons, and an isolectric point of 4.7. By use of a specific radioimmunoassay, it was shown that this glycoprotein is detected only in the epididymis and the deferent duct. It is detectable at 20 days of age and increases progressively to reach a plateau between 3 and 4 months of age (15.7 Â± 0.9 Âµg/mg protein, mean Â± SD, n = 20). It disappears after castration and reappears after testosterone treatment. Estrogens have no effect on its production. lmmunohistochemical studies show that it is produced by epithelial cells of the caput (distal to the initial segment) and of the corpus epididymidis and suggest that its resorption takes place in the cauda epididymidis and the deferent duct.
Replacement of the ovaries of sheep shortly after ovulation with Silastic implants that simulated the physiological patterns and levels of estradiol and progesterone which circulate during the normal estrous cycle produced an "artificial estrous cycle" in which the time course of serum LH and the incidence of estrous behavior closely resembled those of the normal cycle. Neither steroid by itself was effective in this regard. This suggests that two ovarian steroids, estradiol and progesterone, are both necessary and sufficient to account for the secretion of LH and the expression of estrus during the estrous cycle of sheep.
Based on enumeration of maturation-phase spermatids in testicular homogenates from adult men and rats, daily sperm production per gram of testicular parenchyma (DSP/g) was almost seven times greater in rats (21.1 Â± 0.9 x 10 6 vs 3.1 Â± 0.5 x 10 6 ; x Â± SEM, n = 10). The relative inefficiency of the human testis was uniformly expressed in cranial, equatorial and caudal regions. Furthermore, DSP/g was highly correlated between paired testes from individual men (r = +0.97, n = 10), and differences between paired testes were not significantly different from zero (P>0.4). Histometric analysis of glutaraldehyde-perfused testes revealed differences (P<0.05) in relative testicular compositions between humans and rats (n = 3) including higher proportions of parenchyma, seminiferous tubules, seminiferous epithelium and germinal cells in the rat testis. Humans exceeded rats only in the proportions of testis occupied by nongerminal components such as tunic, interstitium, tubule boundary tissue and Sertoli cells. Histometric determination of the proportion of the testicular parenchyma occupied by these nuclei and consideration of average volume of round spermatid nuclei yielded DSP/g estimates of 17.4 Â± 1.8 x 10 6 in rats (not significantly different from the result by the homogenization method) and 8.5 Â± 1.3 x 10 6 in humans (almost three times higher than the value obtained by the homogenization method). Although the disparity between values for human DSP/g obtained by the two methods cannot yet be explained, several lines of evidence suggest that the time divisor used for human material processed by the homogenization method is too long, thus yielding an underestimation of daily sperm production. While its absolute accuracy is questionable, the homogenization method remains a rapid and precise technique for estimating DSP/g in humans. Histometric analysis and histometric estimation of DSP/g support the concept that the human is less efficient in sperm production than the rat, but these methods suggest that the rat is only about twice as efficient per gram of testicular parenchyma as the human.
Serum luteinizing hormone (LH) was measured throughout the afternoon and evening (1500-0130 h) of proestrus in young (4.5 month old) and middle-aged (10.5 month old) female rats displaying regular 4-day estrous cycles as judged by vaginal smears. An LH surge was measured in 22 of 24 young females. In 20 of the young females, the maximum concentration of LH in the blood was measured at 1800 h. The LH surge in middle-aged females was more variable in amplitude, and in 21 of the 29 animals in which a surge was observed it occurred at 1930 h or later. There was a relationship between the time of the surge and its amplitude; "late" surges were consistently lower. These observations demonstrate that marked changes in the pattern of LH secretion occur prior to the time ovarian cyclicity ceases in the aging female rat.
Preparations of Sertoli cells and peritubular myoid cells from testes of 20-day-old rats were cultured alone or together, and cellular interactions were assessed. In the co-cultured system, Sertoli cells within plaques became elevated, forming macroscopically visible mounds and protrusions. The morphologic characteristics of these structures were determined with scanning and transmission electron microscopy. The formation of a limiting membrane observed between individual Sertoli cell nodules and surrounding myoid cells resembled some aspects of the morphology of seminiferous tubules. Long-term co-cultures of Sertoli cells and myoid cells, stimulated by follicle stimulating hormone (FSH), continued to secrete androgen binding protein (ABP) under conditions in which Sertoli cell monocultures ceased production of ABP. The specificity of these cellular interactions was investigated. Bladder smooth muscle cells could substitute for peritubular myoid cells in eliciting mound formation of co-cultured Sertoli cells, but embryonic fibroblasts could not. However, embryonic fibroblasts, which did not secrete ABP, permitted FSH-stimulated Sertoli cells in long-term co-culture to maintain ABP production. In contrast, fibroblasts from the tunica albuginea, when co-cultured with Sertoli cells, neither elicited mound formation nor supported ABP production. We conclude that while unique specificity of cellular interactions between Sertoli cells and peritubular myoid cells is not apparent in cultured preparations from testes of 20-day-old rats, these cells do retain some of the properties required for aggregation and tubule formation characteristic of tubule morphogenesis.
The present work was devoted to the study of certain aspects of the interaction of rat specific epididymal proteins (SEP) with spermatozoa. The amounts of SEP bound to rat sperm from several regions of the epididymis were quantified using a micro-complement fixation technique. Sperm from the caput, corpus, and cauda epididymidis contained increasing amounts of both total SEP and of protein D-E on their surface. Incubations, either in utero or in vitro in conditions similar to those known to favor capacitation, resulted in a substantial loss of SEP from spermatozoa. Exposure of corpus and caudal spermatozoa to solutions containing 0.2 to 0.25 M salt reduced the amount of bound SEP to the level found in caput sperm whereas this treatment had no effect on SEP bound to caput spermatozoa.
Five laying hens were serially sampled via a brachial vein cannula at 2 h intervals for a period of 34 to 72 h. Plasma samples were grouped to the nearest 2 h interval relative to the estimated time of ovulation, and subsequently each sample was assayed for progesterone (P 4 ), estradiol-17Î² (E 2 ), estrone (E 1 ), testosterone (T), 5Î±-dihydrotestosterone (DHT), corticosterone (B), and LH. Mean concentrations of plasma LH, P 4 , E 1 , E 2 , and DHT were highest 6 h prior to ovulation, while a peak of T preceded ovulation by 8 h. There was a surge of B preceding ovulation by 2 h; however, this is attributed to a peak related to oviposition and not to ovulation. There was a small daily surge of LH occurring at the onset of darkness regardless of whether an ovulation occurred on the subsequent morning. These hormone fluctuations are discussed with reference to the potential direct or indirect role of each hormone in the induction of ovulation.
Surface proteins of ram spermatozoa were studied at three stages of post-testicular development: immediately after transport out of the testis; after epididymal transit; and after exposure to accessory sex gland secretions. Testicular and cauda epididymal spermatozoa, together with the fluids which normally surround them, were collected from conscious rams through catheters inserted into the rete testis and vas deferens, respectively. Ejaculated spermatozoa were obtained by electrical stimulation. The washed, viable spermatozoa were radioiodinated by the surface selective hactoperoxidase-glucose oxidase technique. Total sodium dodecyl sulfate-soluble cellular proteins were fractionated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeled surface components were identified and distinguished from unlabeled cellular proteins by autoradiography of dried gel slices. The electrophoretic analysis reveals that the predominant radioiodinated surface components of testicular spermatozoa are seven polypeptides that migrate with apparent molecular weights of 78,000-132,000. Spermatozoa collected from the cauda epididymidis lack the major components characteristic of testicular spermatozoa, but have other unique surface features; these include two large polypeptides (molecular weights, 71,000 and 116,000 daltons) and several smaller components. Most prominent among the latter is a diffuse band of apparent molecular weight 24,000. These striking changes in accessible surface proteins take place during epididymal transit without alterations in the electrophoretic pattern of total cellular proteins. Further surface renovation after spermatozoal maturation is minimal, as shown by the similarity in the radioiodination pattern of ejaculated and cauda epididymal spermatozoa. Since extracellular fluids may play a role in surface modifications of spermatozoa in the epididymis, testicular spermatozoa were incubated in the presence of radioiodinated proteins of rete testis and cauda epididymal fluid. Uptake of labeled protein from rete testis fluid is nonspecific, but adsorption of cauda epididymal fluid proteins by testicular spermatozoa is highly selective. The major adsorbed component migrates with an apparent molecular weight of 24,000 and appears to correspond to the major surface component detectable by radioiodination of cauda epididymal spermatozoa. The anomalous migration of this protein on two-dimensional gel suggests that it is highly glycosylated. These results provide direct biochemical evidence that spermatozoa undergo extensive surface modifications during passage through the epididymis where they become almost totally dependent on exogenous substrates for survival and develop the capacity for coordinated movement. Adsorption of extracellular proteins from the luminal fluids of the testis and epididymis may contribute to these critical functions of the fully differentiated cell.
Movement characteristics of golden hamster spermatozoa within the oviduct at the time and site of fertilization were studied using high-speed cinemicrography. Preparations consisted of excised oviducts immediately submersed in mineral oil and placed in a deep slide-coverglass system at 37Â°C. All spermatozoa visualized were motile and were exhibiting flagellar movement similar to the "activated" motility previously identified by observations in vitro. The geometrical details of this movement were not constant with time, however, and involved episodes of whiplash-like flagellar bending punctuated by periods of more progressive swimming. The trajectories of the spermatozoa were obviously influenced by the presence of the epithelial surfaces of the oviduct, suggesting that interactions between the spermatozoa and these surfaces could play a role in oviductal sperm transport.
Prepubertal gifts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Laparotomies were carried out on groups of four or five gilts 24, 48, 72, 76, 88, 96, 102, or 108 h after PMSG treatment, and follicular fluid (FF) was aspirated from developing follicles. In most cases FF collected from each ovary of individual gilts was pooled, and the concentration of estrone (E 1 ), estradiol-17Î² (E 2 ), androstenedione (A), testosterone + 5Î±-dihydrotestosterone (T+DHT), and progesterone (P) in each sample was determined by radioimmunoassay. The FF concentrations of A and T+DHT remained relatively constant throughout the preovulatory development of the Graafian follicle. The FF concentrations of E 1 , E 2 , and P increased until 72 h after administration of PMSG. Following hCG treatment the FF concentrations of E 1 and E 2 declined rapidly to levels below those observed 24 h after PMSG treatment and remained at these levels, whereas the levels of P remained relatively constant until 30 h after hCG was given and then increased sharply. Resumption of meiosis was first evident by 16 h after injection of hCG and coincided with the decline in FF estrogen concentrations. These results indicate that the microenvironment of the Graafian follicle during preovulatory growth and development undergoes an orderly sequence of changes in steroid hormone concentrations which appear to be modulated by exposure of the developing follicle to gonadotropic stimuli.
Spermatozoa undergo a series of modifications during their transit through the epididymis which lead to the acquisition of fertilizing capacity. The rat epididymis produces and secretes specific proteins (SEP) which interact with the maturing spermatozoa and might be involved in the maturation process. In the present study we used a micro-complement fixation technique to determine the concentration of SEP in the caput, corpus and cauda epididymides and the ratio SEP/spermatozoa in each segment. The highest amounts of SEP were found in the caput and cauda regions (10.42 x 10 3 and 9.69 x 10 3 U.SEP/mg tissue, respectively). The ratio SEP/spermatozoa was highest in the caput (85.5 U.SEP/10 3 spermatozoa) and decreased towards the cauda. The main site of synthesis of SEP was determined after isolation of the different segments and by the de novo synthesis of SEP and found to be the caput region. Immunofluorescence revealed that spermatozoa become coated with SEP as they leave the initial segment and remain coated throughout the organ.
Serum levels of prolactin (PRL), luteinizing hormone (LH), progesterone and estradiol-17Î² were compared in female turkeys during various phases of their reproductive life cycle. Prolactin levels of broody turkeys were higher (2164 Â± 127 ng/ml) than those of laying turkeys (468 Â± 74 ng/ml), which were in turn higher than those of hens that had stopped laying (119 Â± 18 ng/ml). Serum LH, progesterone, and estradiol-17Î² levels of laying hens were significantly higher than those of the other two groups. The levels of these hormones did not differ between broody hens and hens that had stopped laying, but were not broody (photorefractory hens). Serum prolactin levels of broody hens dropped markedly within a day of nest-deprivation and confinement to cages. Levels remained low while hens were in cages. When nests were again made available to broody hens that had been deprived of nests for 48 h, they resumed nesting within 5 min, and serum prolactin levels then increased. In other experiments, it was found that a significant decline in serum prolactin occurred after 8, but not 4 h of nest-deprivation. After a 48 h period of cage confinement of hens, prolactin levels were seen to increase within 30 min after hens were returned to their home pens. Levels continued to rise and had nearly reached pre-cage confinement levels by 12 h after resumption of nesting.
Cow ova and embryos were recovered in 1-16-cell stages and studied by light and electron microscopy. Events associated with normal fertilization and early development in other species were documented by observations of acrosome-reacted sperm cells embedded in the matrix of the zona pellucida, presence of sperm remnants within ooplasm and disappearance of cortical granules, appearance of centrioles at the 8-cell stage, and changes in mitochondria with development. Few sperm cells, apparently limited in penetration to one-third the thickness of the zona, along with complete ovum penetration by only one sperm cell suggested a strong zona block to polyspermy. Sperm remnants were occasionally found in blastomeric cytoplasm of 2-cell stage ova. Prominent granules were seen in mitochondria of bovine ova before and after fertilization. Efforts to achieve fertilization in vitro by combining ova, recovered near the expected time of ovulation from follicles or oviducts, with bull sperm treated with high ionic strength medium resulted in sperm penetration and development to 2- and 4-cell stage embryos judged normal by light and electron microscopy. Use of a different bull was associated with aberrant ovum activation with retention of cortical granules, thus emphasizing a need for further definition of conditions compatible with bovine fertilization.
In ewes, a pivotal neuroendocrine event regulating seasonal breeding is a change in response of the hypothalamo-pituitary axis to the negative feedback action of estradiol. Further, the major environmental parameter regulating seasonal breeding is photoperiod. This study was performed to examine whether changes in photoperiod regulate breeding seasons by modulating response to steroid feedback. Six intact ewes, five estradiol-treated ovariectomized ewes, and one vasectomized ram were housed under artificial short-day (8L:16D) and long-day (16L:8D) photoperiods which were alternated every 90 days. Under these conditions, the intact ewes underwent two breeding and anestrous seasons in 1 year. Further, the transitions between breeding seasons coincided with striking fluctuations in serum LH (between <0.3 and 10 ng/ml) and FSH (50-170 ng/ml) in the estradiol-treated ovariectomized ewes, long days causing anestrus and an increased response to estradiol (low LH and FSH), short days resulting in breeding season and a decreased response to estradiol (high LH and FSH). Similar results were obtained in identical groups of ewes, which were subjected to 120-day alternations between long and short days, except that there were only three seasonal transitions and FSH was not measured. These results provide strong support for the hypothesis that in ewes the mechanism whereby photoperiod regulates seasonal breeding includes modulation of response to the negative feedback action of estradiol on gonadotropin secretion.