Effect of pinealectomy, adrenalectomy, pinealectomy plus adrenalectomy upon the quantification of spermatogenic cells of adult rats

Efeito da pinealectomia, adrenalectomia, pinealectomia mais adrenalectomia sobre a quantificação de células espermatogênicas de ratos adultos

Effect of pinealectomy, adrenalectomy, pinealectomy plus adrenalectomy upon the quantification of spermatogenic cells of adult rats

[Efeito da pinealectomia, adrenalectomia, pinealectomia mais adrenalectomia sobre a quantificação de células espermatogênicas de ratos adultos]

A.C.S. Castro¹, G.F. Queiroz², J.C. Nogueira¹, C.C. Coimbra³, A.M. Reis³ U. Marubayashi³, K. Yamasaki³

¹Departamento de Morfologia do Instituto de Ciências Biológicas da UFMG

Caixa Postal 486

31270-901 - Belo Horizonte, MG

2UEMG/FUNEDI – Campus Divinópolis

3Departamento de Fisiologia e Biofísica do Instituto de Ciências Biológicas da UFMG

Recebido para publicação em 21 de fevereiro de 2002.

E-mail: [e-mail]

ABSTRACT

The objectives of this study were to evaluate the effects of pinealectomy, adrenalectomy and pinealectomy-adrenalectomy upon the quantification of spermatogenic cells of rats. As such, 32 adult Wistar rats with a mean body weight of 331.7± 15.5g were assigned into one of the following treatments: (a) a sham-operated control group, consisting of nine animals; (b) ten pinealectomized animals; (c) seven adrenalectomized animals and (d) six pinealectomized plus adrenalectomized animals. No significant differences were observed between groups for the following parameters: body, testes, prostate and seminal vesicle weights, seminiferous tubular diameter, number of cells per seminiferous tubular cross sections (primary spermatocytes at pachytene, round spermatids, Sertoli cells) and numbers of germ cells per Sertoli cell (primary spermatocytes at pachytene and round spermatids ). Although no increase in testicular weight was observed following pinealectomy, a significant (P<0.05) increase of approximately 11.5% in the number of round spermatids per Sertoli cell (Sertoli cell ratio) occurred thus suggesting that short-term pinealectomy abolishes the antigonadal effect of the pineal gland upon adult Wistar rat testes.

Keywords:Spermatogenesis, pinealectomy, adrenalectomy, rat

RESUMO

Os objetivos deste estudo foram avaliar os efeitos da pinealectomia, da adrenalectomia e da adrenalectomia mais pinealectomia na quantificação das células espermatogênicas de ratos. Assim, 32 ratos adultos Wistar com peso corporal médio de 331,7± 15,5g foram alocados em um dos seguintes tratamentos: (a) um grupo controle simulado, composto de nove animais; (b) dez animais pinealectomizados; (c) sete animais adrenalectomizados e (d) seis animais pinealectomizados+adrenalectomizados. Não foram encontradas diferenças significativas entre grupos para os seguintes parâmetros: pesos corporal, dos testículos, da próstata e das vesículas seminais, diâmetro dos túbulos seminíferos, número de células por corte transversal de túbulo seminífero (espermatócitos primários em paquíteno, espermátides arredondadas, células de Sertoli) e números de células espermáticas por célula de Sertoli (espermatócitos primários em paquíteno e espermátides arredondadas). Apesar do peso testicular não ter aumentado após a pinealectomia, houve aumento significativo (P<0,05) de aproximadamente 11,5% no número de espermátides arredondadas por célula de Sertoli (índice de célula de Sertoli), sugerindo que após curto intervalo a pinealectomia abole o efeito anti-gonádico da pineal sobre os testículos de ratos Wistar adultos.

Palavras-chave: Espermatogênese, pinealectomia, adrenalectomia, rato

INTRODUCTION

The pineal gland has been particularly implicated in the process of mammalian reproduction. However, most studies have centered on the the ability of the pineal gland to influence the hypothalamo-hypophysial-gonadal axis. Although early studies on the pineal function in reproduction have yielded several inconsistencies, probably due to many factors such as species, age, light cycles of the animals, in general, the pineal gland is considered to exert a suppressive action on gonadal maturation and function (Johnson & Reiter, 1978).

Most of what is known concerning this role is derived from experiments employing laboratory rats and golden hamsters. It has proven rather difficult to demonstrate a role for the pineal gland in regulating the reproductive system of rats by the use of pinealectomy. Pinealectomy (PX) of adult rats has been found to cause testicular hypertrophy in some studies (Foa, 1914; Kinel & Bengiano, 1967) and no effects in others (Kitay, 1967; Gorbman et al., 1983). Demaine et al. (1983) have studied the effects of pinealectomy in the spermatogenesis of mature Syrian hamsters housed in either long photoperiods or in continuous darkness and reported a significant reduction in testicular weight and the number of tubules containing sperm in both intact and sham pinealectomized hamsters but not in the pinealectomized animals. These authors suggest that the pineal gland has an antispermatogenic role in animals exposed to longer photoperiods. Adrenalectomy in 30-day-old mice caused a significant decrease in the diameters of seminiferous tubules and Leydig cell nuclei, cell counts of intermediate spermatogonia, round and elongated spermatids (Bhagya & Yajurvedi, 1999).

To our knowledge, studies involving the effects of pinealectomy and adrenalectomy in spermatogenesis of the adult rat are scarce and still controversial. In addition, these studies do not involve the quantitative but rather the qualitative aspects of spermatogenesis. Furthermore, investigations concerning pinealectomy plus adrenalectomy are not yet available in the literature. Therefore, the objectives of this paper are to evaluate the effects of short-term pinealectomy, adrenalectomy and pinealectomy-adrenalectomy in the quantification of some spermatogenic cells of adult Wistar rats under natural light conditions.

MATERIAL AND METHODS

Thirty-two male adult Wistar rats with a mean body weight of 331.7± 15.5g were allocated into one of the following treatments: (a) a sham-operated control group, consisting of nine animals; (b) ten pinealectomized animals; (c) seven adrenalectomized animals and (d) six pinealectomized plus adrenalectomized animals. The surgical procedures employed are described by Yamasaki et al. (1990). Briefly, pinealectomy was performed under pentobarbital anaesthesia by inserting an iredectomy forceps under the confluence of the sagittal and transverse sinuses, breaking the dura mater without invading the sinuses to remove the intact pineal gland. Adrenalectomy was performed by usual the dorsal approach. Completeness of surgical removal of the glands was checked at autopsy. Sham operation was carried out using an identical procedure, except that both the pineal and adrenal glands were not removed. After surgery, the rats were placed in individual cages with free access to Purina chow and Richter tubes with 0.15 M sodium chloride water. Light was provided from 5 a.m. to 7 p.m. each day and temperature was maintained at 25± 1ºC. All animals were killed at 21 days after surgery by decapitation. Testes were immediately removed, weighed and transversally sectioned and the fragments were immersed fixed in Bouin's liquid for 24 hours, dehydrated in increasing concentrations of ethanol, embedded in paraffin, sectioned at 6m; m thickness and stained with hematoxylin-eosin. In addition, the prostate complexes and the seminal vesicles were dissected out and weighed.

Quantitative histological evaluation of the testis included: (a) measurement of seminiferous tubular diameter in ten randomly chosen cross-sections per testis for each animal, using a Zeiss Kpl-w 10x ocular micrometer coupled to a 10x objective and (b) estimation of the seminiferous tubule cell population by counting the nuclei of germ cells (pachytene primary spermatocytes and round spermatids) and of nucleoli of Sertoli cells in ten round cross-sections at stage VII of the seminiferous epithelium cycle (SEC) according to Clermont & Perey (1957). Crude counts were adjusted for nuclear and tubular diameters by the Abercrombie correction factor (Abercrombie, 1946) and the Sertoli cell correction factor (Clermont & Morgenthaler 1955), respectively. For this purpose, an average of ten pachytene primary spermatocytes and round spermatids nuclei and of ten Sertoli cell nucleoli was measured at stage VII of the SEC for each testis with a Zeis Kpl-W 10x micrometer coupled to a 100x objective. Since no significant differences were observed in Sertoli cell nucleoli diameters among groups, a constant Sertoli cell nuclear mean diameter of 2.82m; m± 0.04 was used. Differences between groups were analysed by one-way- Anova with Sigmastat for windows version 1.0 (Jandel Corporation, 1992-1994).

RESULTS

Data for all parameters are summarized in Table 1. As depicted from this table, no significant differences were found among the groups for most parameters analysed (i.e, body weight, testis, prostate and seminal vesicle weights, number of primary spermatocytes, spermatids and Sertoli cell per cross section and number of spermatids per Sertoli cell). However, the number of round spermatids per Sertoli cell increased (P<0.05) approximately 11.5% in pinealectomized versus control rats (6.58 and 5.90, respectively) and 4.5% in adrenalectomized versus control rats (6.17 and 5.90, respectively).

DISCUSSION

Neither body or testes weights, as well as prostate and seminal vesicle were significantly different among treatments in the present study. Furthermore, the relation body to testes weight did not vary across the groups. As also pointed out by Demaine et al. (1983), testicular weight of mature hamsters was not affected by pinealectomy in spite of the longer photoperiod to which these animals had been submitted. Contrary to the hamster, which is a sazonal breeder, the laboratory rat is a continuous breeder and is far less sensitive to lack of photic input. Surgical and environmental manipulations in the rat involving altered pineal activity invariably lead to less dramatic changes in various parameters of male reproductive function (Kinson, 1976).

In adult males, except hamsters, testes and accessory sex organs usually increase in size after pinealectomy, though differential effects are sometimes present between the two groups of organs (Carnicelli et al., 1963; Roth, 1964; Charlton et al., 1976). Roth (1964) found no effects of pinealectomy on testicular weight in rats, but found increases in seminal vesicle and prostate weights.In the present investigation, no significant differences were observed in accessory sex organs weight among groups, which might be expected since no corresponding increase in testes weight was noted.

No variations in the seminiferous tubular diameters was found among groups in the present study. Likewise, adult pinealectomized Syrian hamsters exhibited seminiferous tubular diameters similar to those intact animals (Demaine et al.,1983). However, adrenalectomy in 30 day old mice caused a significant decrease in the diameters of seminiferous tubules (Bhagya & Yajurvedi, 1999). Nair et al. (1995) reported degenerative changes such as decreased seminiferous tubule diameter, Leydig cell nuclear diameter, spermatogenic arrest, oedematous fluid in the interstitium and lumen of the seminiferous tubules of Sprague-Dawley rats weighing 290-350g and sacrificed at 2, 4, 6, 8 and 16 days after adrenalectomy. The discrepancy between our findings and those of Bhagya & Yajurvedi (1999) and Nair et al. (1995) could probably be attributed to species differences (mice vs rats) and to the different interval from adrenalectomy to sacrifice.

In the present study, the numbers of germ cells (i.e, primary spermatocytes at pachytene and round spermatids) as well as of Sertoli cells per tubular cross section between control and treated groups did not vary. However, the number of round spermatids per Sertoli cell (Sertoli cell ratio) was significantly different between the control and the pinealectomized groups. In fact, an increase of approximately 11.5% in the number of these cells was seen in the pinealectomized animals as compared to the controls (6.58vs5.90 spermatids/Sertoli cell, respectively). Interestingly, no such significant difference was observed in the number of primary spermatocytes per Sertoli cell in the same two groups, in spite of the apparent increase of about 9% in the number of spermatocytes per Sertoli cell in pinealectomized animals. It is worthy of mention that one cycle of the seminiferous epithelium cycle lasts approximately 13.3 days (Huckins, 1965) while the entire process of spermatogenesis is equivalent to 4.5 cycles (Berndtson, 1977). Therefore, since the time from pinealectomy to sacrifice (21 days) corresponds to aproximately two cycles of the seminiferous epithelium, it is reasonable tospeculate that had this interval been greater (i.e., about 60 days), it is possible that the increase in the spermatogenic cell numbers might have been greater and have included cells at the earlier stages of spermatogenic process (e.g; pachytene spermatocytes).Although in an abstract form, Demaine et al. (1983)also reported an increase in the number of spermatogenic cells (although no specific cell type was indicated) per unit area of testicular tissue irrespective of the lighting conditions to which the pinealectomized Syrian hamsters had been exposed, suggesting that the pineal gland has an antispermatogenic role in animals exposed to longer photoperiods. In spite of the species differences, our results are also indicative of this antispermatogenic action exerted by the pineal gland. Grandi (1983) reported that the testicular parenchyma of 60-80 days-old albino rats weighing 260-280g showed morphological aspects of hypotrophy characterized by the absence of spermatozoa in tubular lumen and by tubules lined by an immature seminiferous epithelium after 8 and 16 days from total pinealectomy characterized by an apparent reduction of some germ cell types and by the 21^st day, spermatogenesis had been restored to normal. Grandi's findings are suggestive of a participation of the pineal in the temporal regulation spermatogenesis in the albino rat. Although no quantitative data was provided in Grandi's study, it is possible that since the interval from pinealectomy to sacrifice in our study was that of 21 days, it is likely that spermatogenesis of Wistar rats might have been restored by this time. However, this does not yet explain the increase in the number of spermatids observed in the present study.

Contrary to the findings of Baghya & Yajurvedi (1999) who reported reduced Leydig cell nuclear and seminiferous tubular diameters and a reduction in cell counts (intermediate spermatogonia and elongated spermatids) of 30-day-old adrenalectomized mice, no such reduction in the number of round spermatids per Sertoli cell was observed (P>0.05) in adrenalectomized versus control adult rats in the present study. There is no apparent explanation for the discrepancy between our findings and those of Baghya & Yajurvedi (1999) but possible factors might be atributted to species (mice versus rats) and age (young mice versus adult rats) differences, as well as differences in methodology employed.

All biometric (body, prostate, seminal vesicle and testes weights) as well as the histologic (seminiferous tubular diameter, number of testicular cells per seminiferous cross section and number of cells per Seroli cell) parameters of the pinealectomized plus adrenalectomized group showed no significant differences from the control group, thus indicating that adrenalectomy in association with pinealectomy apparently does not impair the process of spermatogenesis.

In conclusion, pinealectomy caused a significant increase in the number of round spermatids of adult Wistar rats, which is indicative of the antigonadal role exerted by the pineal. However, further studies involving both different time intervals (short and long) from pinealectomy and adrenalectomy to sacrifice and the quantitative aspects of the various cell types within the testis are fundamental for a better understanding of the dynamics of spermatogenesis of the rat under these adverse circunstances.

ACKNOWLEDGMENTS

This study was financially supported by CNPq (Brasilia) and FAPEMIG (Belo Horizonte), Brazil.

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Publication Dates

• Publication in this collection
  16 Dec 2002
• Date of issue
  June 2002

History

• Received
  21 Feb 2002