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doI 10.11603/IJMMR.2413-6077.2020.1.11502

MORPHOMETRIC EVALUATION OF SPERMATOGENIC EPITHELIOCYTES
STRUCTURE UNDER THE RUBOMYCIN INFLUENCE IN RATS

M.S. Hnatiuk, *S.О. Konovalenko, L.V. Tatarchuk, О.B. Yasinovsky
I. HORBACHEVSKY TERNOPIL NATIONAL MEDICAL UNIVERSITY, TERNOPIL, UKRAINE

Background. Over the last 20 years, the quality of men’s sperm around the world has deteriorated twice.
The causes are poor environment, stress, sedentary lifestyle, prevalence of sexually transmitted infections, obesity,
alcohol abuse and various stimulants. The structure and function of the testes, when exposed to various drugs
and chemicals, attracts the attention of present researchers.

Objectives. The aim of the research was to study the structural changes of the spermatogenic epithelium
in testicular damage caused by rubomycin hydrochloride.

Methods. The spermatogenic epithelium of the testes of 30 white adult white male rats, which were divided
into 2 groups, was morphometrically examined. The 1st group comprised 15 experimental intact animals, the 2nd
– 15 rats, in which testicular damage with rubomycin hydrochloride was simulated. Euthanasia of rats was
performed by bloodletting under thiopental anesthesia one month after the experiment beginning. Quantitative
morphological parameters were statistically processed. In spermatocytes of the 1st P-order, spermatogonia and
spermatids of testes, their height, diameter of nuclei, nuclear-cytoplasmic ratio and relative volumes of damaged
cells of spermatogenic epithelium were evaluated.

Results. It was established that spermatogenic epithelial cells were damaged in the simulated experimental
conditions, which was morphometrically confirmed by changes in nuclear-cytoplasmic relations in the 1st, 2nd
order spermatocytes, spermatogonia and spermatids. The relative volumes of damaged spermatogenic epithelial
cells also increased significantly. The most significant morphometric parameters were altered in spermatids.
Thus, the nuclear-cytoplasmic ratio in the studied cells was statistically significantly (p <0.001) increased by 7.4%
compared with the similar control morphometric parameter. The relative volume of damaged spermatids in these
experimental conditions reached (32.50±0.18) %, which with a high statistically significant difference (p<0.001)
exceeded the same control value in almost 15.8 times. Optically, histological preparations of the testes showed
severe vascular disorders, characterized by dilation and plethora mostly of venous vessels, which pointed to
violation of venous drainage of the studied organs.

Conclusions. According to the attained research results it has been established that nuclear-cytoplasmic
relations are an objective and valuable informative indicator of a functional condition of cells and their structural
changes in pathological conditions. When rubomycin hydrochloride is exposed to experimental animals, the most
significant changes in nuclear-cytoplasmic relations are found in spermatic spermatozoa.

KEY WORDS: rubomycin hydrochloride, testes, spermatogenic epithelium, white rats,
morphometry.

International Journal of Medicine and Medical Research
2020, Volume 6, Issue 1, p. 68-73
copyright © 2020, TNMU, All Rights Reserved

*Corresponding author: S.О. Konovalenko, MD, Ph.D., Associate
Professor, Department of Operative Surgery and Clinical
Anatomy, I. Horbachevsky Ternopil National Medical Univer-
sity, Ternopil, Ukraine; e-mail: konovalenko @tdmu.edu.ua

M.S. Hnatiuk et al.

Introduction
It is established that in the structure of

infertile marriages, the male factor is 20% [2, 4,
8, 12, 17]. The reason for this is a decrease in
spermatogenic and hormonal function of the
testes. Often the reason for this is the effect of
various toxic factors on the body [2, 13, 14, 19].
In recent years, many researchers are interested
in changes in the structure and function of the
testes in different physiological and pathological
conditions [10, 11, 15, 19].

Rubomycin is an antitumor antibiotic of the
anthracycline series with a significant cytostatic

effect, which when administered to men can
lead to azoospermia (absence of sperm in the
ejaculate) [5]. The effect of rubomycin on the
structure of the spermatogenic epithelium has
been studied insufficiently.

The aim of our study was to investigate the
features of structural changes in the sperma-
to genic epithelium in cases of testicular da-
mage by rubomycin hydrochloride.

Methods
The testicular structures of 30 laboratory

adult white male rats, which were divided into
2 groups, were studied by morphometric me-
thods. The 1st group comprised 15 exper mental
intact animals, the 2nd – 15 rats with simulated

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iSSn 2413-6077. iJmmR 2020 Vol. 6 issue 1 M.S. Hnatiuk et al.

testicular damage by rubomycin hydrochloride
[18]. One month after the beginning of the
experiment, euthanasia of experimental ani-
mals was performed by bloodletting under
conditions of thiopental-sodium anesthesia.
Pieces were cut from the testes, which were
fixed in 10 % neutral formalin solution, passed
through ethyl alcohols of increasing concent-
ration and placed in paraffin. Microtome sec-
tions 5-6 mcr thick after dewaxing were stained
with hematoxylin-eosin, according to van Gizon,
Mallory, Weigert, toluidine blue [7]. Histological
micropreparations determined the height of
spermatogenic cells, the diameter of their
nuclei, nuclear-cytoplasmic ratios, the relative
volume of damaged epitheliocytes (VODE) [1,
6, 16]. Morphometry of these structures was
performed using a light microscope “Olimpus
BX-2” with a digital video camera and application
package “Video Test 5.0” and “Video size 5.0”.
Quantitative values were processed statistically
by STATISTIKA software package. The diffe-
rences between the comparative values were
determined by the Mann-Whitney and Student’s
tests [9]. The studies were performed according
to national and international regulations on
carrying out experimental tests (“European
Convention for the Protection of Vertebrate
Ani mals used for Experimental and Other Scien-
tific Purposes” (Strasbourg, 1986), “General
Ethical Principles of Animal Experiments”,
adop ted by the First National Congress on Bio-
ethics (Kiev, 2001)) [8], as well as the Law of

Ukraine “On Protection of Animals from Cruel-
ty”, dated February 21, 2006.

Results
The attained morphometric parameters are

presented in Table 1. A comprehensive analysis
of the data showed that most of the studied
morphometric parameters of the spermatogenic
epithelium changed significantly. Thus, the
height of spermatocytes of the 1st order de-
creased slightly by only 0.65% (p<0.05). The
diameters of the nuclei of these cells with a high
statistically significant difference (p<0.001)
increased from (5.25±0.03) µm to (5.76±0.04)
µm, i.e. by 9.7%. Uneven changes in the spatial
characteristics of the nucleus and cytoplasm of
spermatocytes of the 1st order led to violations
of nuclear-cytoplasmic relations in them. This
morphometric index increased by 3.2% (p<0.05),
which pointed to violation of the studied ele-
ments of structural cellular homeostasis [1].
The relative volume of damaged first-order
sper matocytes under these experimental con-
ditions with a high statistically significant diffe-
rence (p<0.001) increased almost in 9.7 times.

The morphometric parameters of P-order
spermatocytes changed almost similarly under
the influence of rubomycin hydrochloride. The
height of these cells was almost the same as in
the control, the diameters of the nuclei were
statistically significantly (p<0.05) increased by
2.4%, and the nuclear-cytoplasmic ratio – by
4.9% (p<0.01). The relative volume of damaged

Table 1. Morphometric characteristics of cells of spermatogenic epithelium of testes of the
experimental animals (M±m)

Cells
Morphometric parameter

Height, microns Diameter of the core, microns
Nuclear-cytoplas-

mic relations VODE, %

Control group
Spermatocytes of the first
order

7.65±0.05 5.25±0.03 0.556±0.003 2.20±0.01

Spermatocytes of the
second order

5.88±0.04 3.76±0.03 0.410±0.002 2.26±0.02

Spermatogonia 6.12±0.04 4.80±0.03 0.615±0.004 2.15±0.02
Spermatids 4.98±0.03 3.07±0.03 0.389±0.002 2.06±0.02

Experimental group
Spermatocytes of the first
order

7.60±0.05 5.76±0.04*** 0.574±0.004* 21.30±0.15***

Spermatocytes of the
second order

5.85±0.04 3.85±0.03* 0.430±0.003** 25.60±0,15***

Spermatogonia 6.05±0.04 4.79±0.03 0.628±0.004* 29.80±0.15***
Spermatids 4.92±0.03 3.18±0.02* 0.418±0.002*** 32.50±0.18***

Notes. * – р<0.05; ** – р<0.01; *** – р<0.001. VODE – volume of damaged epitheliocytes.

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P-order spermatocytes under these experi-
mental conditions was equal to (25.60±0.15)%.
This morphometric parameter with a high
statistically significant (p<0.001) difference
exceeded the similar quantitative morphological
index in 11.3 times.

The height of spermatogonia of the testes
in the experimental animals affected by the
studied drug medium decreased by only 1.1%
(p<0.05), the diameters of their nuclei did not
change significantly, and nuclear-cytoplasmic
ratios increased by 2.1% (p<0.05). The relative
volume of damaged spermatogonia in these
experimental conditions statistically signi fi-
cantly (p<0.001) increased in 13.8 times and
reached (29.80±0.15)%.

Analysis of the obtained data revealed that
under the influence of rubomycin hydrochloride
the height of testicular spermatids decreased
by only 1.2% (p<0.05), the diameters of their
nuclei increased by 3.6% (p<0.05). Nuclear-
cytoplasmic ratios in the studied cells statistically
significantly (p<0.001) increased by 7.4% com-
pared with the similar control morphometric
parameter. The relative volume of damaged
spermatids under these experimental condi-
tions reached (32.50±0.18) %. This morphometric
parameter with a high statistically significant
difference (p<0.001) exceeded the similar
control value in almost 15.8 times.

Discussion
Analysis of the above morphometric para-

meters proved that the introduction of rubo-
mycin hydrochloride into the body of ex peri-
mental animals led to structural rearrangement
and damage to cells of spermatogenic epithe-
lium of the testes. The most pronounced
changes were found in spermatids in com-
parison with other studied cells.

According to present researchers, nuclear-
cytoplasmic relations are a valuable morpho-
metric indicator that adequately reflects the
peculiarities of cell function and metabolism,
degree of disturbances of cellular structural
homeostasis [1, 6, 16]. The nucleus and cyto-
plasm of cells are separated from each other,
but at the same time they are closely inter-
connected and integrated and form a single
structural and functional system. It follows that
the isolated study of quantitative morphological
parameters of only the cytoplasm of the cell or
only its nucleus allows getting a one-sided idea
of these structural components of the cell. The
study of nuclear-cytoplasmic relationships
makes it possible to obtain more adequate,

objective and in-depth information on the
relationship between the nucleus and cytoplasm
of cells and their changes in various physiological
and pathological conditions. Nuclear-cyto-
plasmic relations can change at hyperfunction
of bodies as well as hypofunction, and also in
cases of their damage. It is established that
nuclear-cytoplasmic ratios depend on cell diffe-
rentiation. The specified morphometric para-
meter decreases in case of maturation of cells
and their differentiation that is caused to some
extent by increase in cytoplasm and its hyper-
plasia of specific functioning organelles. The
relationship between the spatial charac teristics
of the nucleus and cytoplasm of the cell can
also change with cell division, growth, diploidy
[6, 16].

In the optical examination of testicular
micro preparations, significant vascular disor-
ders were evidenced, which were characterized
by dilation and plethora of mainly venous
vessels, which pointed to violation of venous
drainage from the studied organs (Fig. 1) [3].

A severe edema of the stroma, foci of
dystrophically, necrobiotically altered sperma-
togenic epitheliocytes, stromal structures, local
cellular infiltrates, sclerotic processes, desqua-
mation and proliferation of vascular endothelial
cells was present (Pic. 2). The latter pointed to
the presence of hypoxia. Foci of vascular en do-
thelial cell edema, permeation of their memb-
rane with plasma proteins were also present.
Foci of fibrinoid swelling and necrosis in some
vessels were evidenced that indicated their
severe damage. Among spermatogenic epithe-
liocytes in the studied conditions of the expe-
riment, spermatids were the most morpho lo-
gically altered, in which the degree of disorders

Fig. 1. Stromal edema, partial reduction of layers and
damage of spermatogenic epithelial cells, their des-
quamation, thickening of the membranes of the
tortuous seminal tubules of the testis of a white rat
under the influence of rubomycin hydrochloride.
Hematoxylin-eosin staining. ×140.

M.S. Hnatiuk et al.

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iSSn 2413-6077. iJmmR 2020 Vol. 6 issue 1

of nuclear-cytoplasmic relations and the rela-
tive volume of damaged cells were the largest.
It points to the fact that the studied mor-
phometric parameter also reflects the severity
and depth of cell damage.

Conclusions
Nuclear-cytoplasmic relations are an objec-

tive and significant informative indicator of
functional state of the cells and their structural
changes in pathological conditions. When rubo-
mycin hydrochloride is exposed to experimental
animals, the most significant changes in nu-
clear-cytoplasmic relations are found in sper-
matic spermatozoa.

Funding
This research received no external funding.
Conflict of Interests
The authors declare no conflict of interest.
Author Contributions
Hnatiuk M.S. – supervision, morphometry,

morphological analysis of micropreparations,
writing – review and editing; Konovalenko S.О. –
writing, generalization of results, project admi-
nistration, Tatarchuk L.V. – supervision, review
and editing , Yasinovsky О.B. – literature analysis,
data curation.

Fig. 2. Structural changes in the vessel wall, perivasal
sclerosis, dystrophy, edema, desquamation of sper-
matogenic epitheliocytes in the tortuous seminal
tubules of the testis of experimental animals under
the influence of rubomycin hydrochloride. Hematox-
ylin-eosin staining. ×140.

МОРФОМЕТРИЧНА ОЦІНКА СТРУКТУРНОЇ ПЕРЕБУДОВИ КЛІТИН
СПЕРМАТОГЕННИХ ЕПІТЕЛІОЦИТІВ СІМ′ЯНИКІВ ЕКСПЕРИМЕНТАЛЬНИХ
ТВАРИН ПІД ВПЛИВОМ РУБОМІЦИНУ ГІДРОХЛОРИДУ

М.С. Гнатюк, С.О. Коноваленко, Л.В. Татарчук, О.Б. Ясіновський
ТЕРНОПІЛЬСЬКИЙ НАЦІОНАЛЬНИЙ МЕДИЧНИЙ УНІВЕРСИТЕТ ІМЕНІ І.Я.ГОРБАЧЕВСЬКОГО,

ТЕРНОПІЛЬ, УКРАЇНА

Вступ. За останні 20 років якість сперми чоловіків у всьому світі погіршилась у 2 рази. Причини:
погана екологія, стреси, малорухливий спосіб життя, поширеність статевих інфекцій, ожиріння,
зловживання алкоголем і різними стимуляторами. Структура та функція сім′яників при дії на організм
різних медикаментозних середників та хімічних речовин привертає увагу сучасних дослідників.

Мета. Вивчити особливості структурних змін сперматогенного епітелію при ушкодженні сім’яників
рубоміцином гідрохлориду.

Методи. Морфометрично досліджено сперматогенний епітелій сім′яників 30 білих статевозрілих
білих щурів-самців, які були були поділені на 2-і групи. 1-а група включала 15 дослідних інтактних тварин,
2-а – 15 щурів, у яких моделювали ушкодження сім′яників рубоміцином гідрохлоридом. Евтаназію щурів
здійснювали кровопусканням в умовах тіопенталового наркозу через місяць від початку досліду. Кількісні
морфологічні показники обробляли статистично. У сперматоцитах 1-го. П-го порядків, сперматогоніях
та сперматидах сім′яників вимірювали їх висоту, діаметр ядер, ядерно-цитоплазматичні відношення
та відносні об′єми пошкоджених клітин сперматогенного епітелію.

Результати. Встановлено, що у змодельованих експериментальних умовах пошкоджувалися клітини
сперматогенного епітелію, що морфометрично підтверджувалося змінами ядерно-цитоплазматичних
відношень у сперматоцитах 1-го, 2-го порядків, сперматогоніях та сперматидах. Виражено зростали
також відносні об′єми пошкоджених вказаних клітин сперматогенного епітелію. Найбільш виражено
морфометричні параметри виявилися зміненими у сперматидах. Так, ядерно-цитоплазматичні
відношення у досліджуваних клітинах статистично достовірно (р<0,001) збільшилися на 7,4 % порівняно
з аналогічним контрольним морфометричним параметром. Відносний об′єм пошкоджених сперматид
у даних умовах експерименту досягав (32,50±0,18) %, що з вираженою статистично достовірною
різницею (р<0,001) перевищував аналогічну контрольну величину майже у 15,8 рази. Світлооптично
на гістологічних препаратах сім'яників cпостерігалися виражені судинні розлади, що характеризувалися

M.S. Hnatiuk et al.

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розширенням та повнокров′ям переважно венозних судин, що свідчило про порушення венозного дренажу
від досліджуваних органів.

Висновки. На основі проведених досліджень та отриманих результатів встановлено, що ядерно-
цитоплазматичні відношення є об′єктивним та цінним інформативним показником функціонального
стану клітин та їх структурних змін в патологічних умовах. При дії на організм дослідних тварин
рубоміцину гідрохлориду найвираженіші зміни ядерно-цитоплазматичних відношень виявлені у
сперматидах сім′яників.

КЛЮЧОВІ СЛОВА: рубоміцин гідрохлорид, сім′яники, сперматогенний епітелій, білі щурі,
морфометрія.

Інформація про авторів
Гнатюк Михайло Степанович – доктор медичних наук, професор, завідувач кафедри оперативної

хірургії та клінічної анатомії, Тернопільський національний медичний університет імені І.Я. Горба-
чевського.

ORCID http://orcid.org/0000-0002-4110-5568, e-mail: hnatjuk@tdmu.edu.ua
Коноваленко Сергій Олександрович – канд. мед. наук, доцент кафедри оперативної хірургії та

клінічної анатомії, Тернопільський національний медичний університет імені І.Я. Горбачевського.
ORCID http://orcid.org/0000-0002-3478-462X, e-mail: konovalenko @tdmu.edu.ua
Татарчук Людмила Василівна – доктор медичних наук, асистент кафедри фізіології з основами

біоетики та біобезпеки, Тернопільський національний медичний університет імені І.Я. Горбачевського.
ORCID http://orcid.org/0000-0002-4678-4205, e-mail: tatarchyklv@tdmu.edu.ua
Ясіновський Олег Борисович – канд. мед. наук, асистент кафедри оперативної хірургії та клінічної

анатомії, Тернопільський національний медичний університет імені І.Я. Горбачевського.
ORCID http://orcid.org/0000-0002-5121-3140, e-mail: yasinovskyi@tdmu.edu.ua

Information about the authors
Hnatjuk M.S. – MD, Ph.D., DSc, Professor, Head of the Department of Operative Surgery and Clinical

Anatomy, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine.
ORCID http://orcid.org/0000-0002-4110-5568, e-mail: hnatjuk@tdmu.edu.ua
Konovaleno S.О. – MD, Ph.D., Associate Professor, Department of Operative Surgery and Clinical

Anatomy, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine.
ORCID http://orcid.org/0000-0002-3478-462X, e-mail: konovalenko @tdmu.edu.ua
Tatarchuk L.V. – MD, Ph.D., DSс, Assistant Professor, Department of Operative Surgery and Clinical

Anatomy, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine.
ORCID http://orcid.org/0000-0002-4678-4205, e-mail: tatarchyklv@tdmu.edu.ua
Yasinovsky О.B. – MD, Ph.D., Assistant Professor, Department of Operative Surgery and Clinical

Anatomy, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine.
ORCID http://orcid.org/0000-0002-5121-3140, e-mail: yasinovskyi@tdmu.edu.ua

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Received 26 Mar 2020; revised 07 Apr 2020;
accepted 29 May 2020.

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M.S. Hnatiuk et al.