Publisher Summary This chapter focuses on the seasonal control of fertility. It discusses two aspects in detail—the way in which changes in the secretion of luteinizing hormone-releasing hormone by the hypothalamus controls the activity of the pituitary and testis, and the way changes in day length influence the activity of the hypothalamus. The downstream events evoked by the hypothalamus are relatively easy to understand, whereas the mechanisms involved in the photoperiodic control are very complex and largely unresolved. While seasonal changes in temperature, rainfall, and food availability are the factors of the environment that dictate survival of adults and young—and are thus ultimately responsible for dictating the timing of the birth season—these are not necessarily the factors used as cues by the animals to regulate their reproductive endocrinology. This is because it is necessary to anticipate the timing of birth by dictating the timing of conception, as the duration of gestation in mammals is usually fixed. As accurate timing of conception is important, animals tend to become reliant on cues from the environment, which are the best predictors of the time of year. The seasonal cycle highlights the seasonal changes in daylight length, rutting behavior, testicular diameter, sexual skin flush, and concentrations of plasma follicle-stimulating hormone, prolactin, and testosterone in a group of rams throughout the year.

Seasonal breeding:nature's contraceptive.

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.

Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Neuropeptide Y: role in light-dark cycle entrainment of hamster circadian rhythms

Much of the work of the pathologist in understanding male reproductive toxicity comes from trying to understand the initial site of toxicant action – that is, distinguishing the initial effects from the follow-on events. These changes can happen because of, or completely independent of, initial impacts on male reproductive hormones. Fortunately, the patterns of response and the holistic evaluation of a wide variety of different but complementary data allow for considerable confidence in assigning an initiating site of action. This chapter will review the anatomy and physiology as well as successful strategies for understanding toxicities in this complicated and sublime multi-organ system.

Male Reproductive System

This chapter discusses the modulatory influences regulating the timing of puberty in rats. The developmental process that leads to puberty in the female rat is based on an extraordinarily complex series of interrelated events. The central nervous system (CNS) plays a critical role by controlling both anterior pituitary function, through the secretion of hypothalamic factors, and the ovary via pituitary hormones and direct neural inputs. The central neuroendocrine regulation of luteinizing hormone-releasing hormone (LHRH) production and gonadotropin secretion initiates the onset of puberty in rats through LH actions on the Leydig cells to increase androgen production and follicle-stimulating hormone (FSH) actions on the Sertoli cell to enhance Leydig cell function and initiate Sertoli cell differentiation needed for the induction of spermatogenesis. The negative feedback of androgens and inhibin regulate gonadotropin production during the progression of puberty to the adult stage. The coordinated control of this hypothalamic–pituitary axis with the testis involves molecular events at the LHRH neuron and pituitary levels and at the testis somatic cell level. The resulting endocrine events control the onset and progression of puberty in the rat.

CHAPTER 38 – Puberty in the Rat

We studied the temporal aspects of endocrine signaling between the pituitary gland and testes by measuring moment to moment changes in blood LH and testosterone levels in individual male rats. Each rat was fitted with an indwelling vascular cannula, and blood was withdrawn every 5 min for 8–12 h. Rats were maintained throughout the intensive blood-sampling period with an isotonic blood replacement mixture containing rat red blood cells and a human plasma protein preparation. LH and testosterone measurements were made in plasma volumes of 50 and 60 μl. Most rats released LH in well defined pulses, characterized by a rapid increase in plasma LH within 5–10 min and a gradual decline lasting for the next 50–70 min. LH pulses occurred singly or in trains of two to four. Episodes of testosterone secretion spanned 3–6 h and were marked by a slowly graded rise and fall of plasma testosterone. In several instances, testosterone episodes were preceded (1–2 h) by a train of closely coupled LH pulses. Within a partic...

Male Rats Secrete Luteinizing Hormone and Testosterone Episodically

We compared the effects of light pulses in constant darkness (DD) and dark pulses in constant light (LL) on the free-running rhythm of locomotor activity in male golden hamsters. Light pulses yielded advances, delays, or no change in the rhythm of activity. These data conform to a typical phase-response curve; this curve was unaffected by pinealectomy. Dark pulses occurring either late in the subjective night or early in the subjective day had little effect. In contrast, dark pulses occurring either late in the subjective day or early in the subjective night altered the rhythm in one of three ways: advance of the rhythm; splitting into two components; or induction of a new component, in phase with the pulse. Because dark pulses in LL perturb the circadian system in a different manner than do light pulses in DD, they may have value in identifying heretofore unknown aspects of circadian systems. As such, the use of dark pulses to perturb circadian rhythmicity will be a useful tool in examining the formal properties of circadian systems.

Dark pulses affect the circadian rhythm of activity in hamsters kept in constant light

Distinct, short-term pulses of pituitary luteinizing hormone (LH) release are a characteristic feature of tonic LH secretion in normal rats. LH pulses are more pronounced, more frequent, and more regular in castrated rats. Using castrated rats, we sought to identify the basis of the pulsatile discharge of LH by the pituitary gland. Indwelling atrial cannulae were used to obtain frequent blood samples through 3-4 h from three cohorts of conscious, freely moving, orchidectomized male rats. Rats were (1) untreated castrates, (2) infused with ovine antiserum to LHRH or control serum, or (3) infused with a luteinizing hormone releasing hormone (LH-RH) analog, [D-pGlu1,D-Phe2, D-Trp3,6]-LH-RH. Castrates exhibited a pulsatile pattern of circulating LH levels; the mean (+/- SE) peak level of LH pulses was 640 +/- 15 ng/ml, with a mean (+/- SE) pulse period of 18.5 +/- 0.8 min. LH-RH antiserum arrested pulsatile LH secretion immediately, leaving plasma LH levels at 80-120 ng/ml. The LH-RH analog caused a similar suppression of LH release, although this effect was of a shorter duration than the suppression of LH pulses induced by LH-RH antiserum. The obliteration of LH pulses by anti-LH-RH and suppression of LH release by an LH-RH antagonist indicate that the pulsatile secretion of LH is due to corresponding stimulation of the pituitary gland by hypothalamic LH-RH. Anti-LH-RH and an LH-RH antagonist are identified as valuable probes for the experimental dissection of blood-borne signals within the rat hypothalamic-pituitary-testicular axis.

Control of pulsatile LH release in male rats.

ABSTRACT. Exposure to short days [6 h of light/24 h (LD 6: 18)] for 10 weeks renders the hypothalamic-pituitary axis of the castrated male golden hamster extremely sensitive to the negative feedback effect of testosterone. The present study investigated the time course of this change in sensitivity to steroid feedback, in both animals shifted from a stimulatory to a nonstimulatory photoperiod and animals shifted from a nonstimulatory to a stimulatory photoperiod. Hamsters were castrated and maintained on LD 14:10 for 60 days and then implanted sc with either empty or testosteronefilled Silastic capsules that were 4, 8, or 20 mm long. Half of theanimals remained on LD 14:10 and half were transferred to LD 6:18. In animals maintained on LD 14:10, the 4-mm testosterone implants had no effect on serum levels of LH and FSH, while the larger doses of testosterone induced a reduction in serum gonadotropin levels. The effect of testosterone on serum gonadotropin levels in LD 14:10 animals did not vary from 10 day...

Time course of the photoperiod-induced change in sensitivity of the hypothalamic-pituitary axis to testosterone feedback

We charted the development of pulsatile luteinizing hormone (LH) secretion as a function of the time elapsed after removal of the testes. On seven occasions between the moment of castration and 80 days afterwards, we obtained consecutive blood samples at frequent (2.5- to 5-min) intervals from cannulated male rats. Orchidectomy increased both the amplitude and frequency of LH release within 1 day after surgery. Amplitude: From 19 h through 80 days postcastration, peak LH levels rose steadily, and LH pulses grew progressively more pronounced in nadir-to-peak amplitude. Frequency: Our findings offer new evidence establishing an increase in LH pulse frequency from less than 1 per h to 2-3 per h within 1 day after orchidectomy. Once deprived of testicular influences, the frequency of pulsatile LH discharges remained static through 80 days. The sudden onset (less than 1 day after castration) and temporal uniformity of high-frequency LH pulses demonstrate that LH release is governed by an intrinsic, 20- to 30-min neural periodicity in castrate rats. Most important, these findings imply that the testes mask or modulate the expression of an intrinsic, 20- to 30-min neural generator directing the periodic discharge of LH in the intact male rat.

Gary B. Ellis

Papers

Male Rats Secrete Luteinizing Hormone and Testosterone Episodically

Dark pulses affect the circadian rhythm of activity in hamsters kept in constant light

Control of pulsatile LH release in male rats.

Time course of the photoperiod-induced change in sensitivity of the hypothalamic-pituitary axis to testosterone feedback

Orchidectomy Unleashes Pulsatile Luteinizing Hormone Secretion in the Rat