Female annual reproductive cycle of Rhinolophus ferrumequinum korai (Chiroptera: Rhinolophidae): morphological changes and prolonged sperm storage and sperm fate of the female reproductive tract according to month

Abstract

Morphological changes and prolonged sperm storage and loss of female reproductive tract per year and site as well as reproductive cycle were investigated by using an optical microscope, scanning electron microscope and transmission electron microscope in Rhinolophus ferrumequinum korai. The size and morphological characteristics of the uterus were found to be highly relevant to mating and the delivery period. In particular, some of the sperm ejaculated to the female reproductive tract became extinct due to phagocytosis by leukocytes starting from the end of August, which continued until the end of February. Accordingly, this period may be called the cleansing period. The sperm became extinct mainly in the uterine lumen, and an extremely small amount of sperm became extinct by secretion from the uterine gland and the phagocytosis by leukocytes. Such phagocytosis seemed to result in maintaining the clean intrauterine environment to facilitate the implantation of the fertilized egg. Also, phagocytosis by leukocytes was considered as a physiological mechanism to kill the sperm breaking into the female reproductive tract by recognizing them as alien materials, to maintain an appropriate number of sperm at the fertilization site of the fallopian tubes, so as to improve fertility. Only the sperm reaching the caudal isthmus of oviduct were found to be involved in fertilization in the next year. Ovulation and fertilization occurred approximately at the end of March. Embryogenesis appeared consecutively with implantation. The pregnancy period was approximately 70 days; delivery occurred approximately early- and middle June; and the nurturing period was approximately 80 days.

Keywords:

• female annual reproductive cycle
• cleansing period
• prolonged sperm storage
• sperm fate
• phagocytosis

Introduction

In terms of animal reproductive pattern, a number of mammal species subject to seasonal reproduction react to annual climate change (Deshmukh & Dhamani 2011). In some bats, foodstuffs and ambient temperature impact the embryonic development and gestation period (Racey 1973), and major factors impacting the reproductive cycle include hormone (Kawamoto 2003) as well as a number of environmental changes such as diet, water, habitat space, climate condition and availability of climate (Vasantha 2016).

The male reproductive cycle of hibernating bats is classified as three types, “Pipistrellus pattern”, “Myotis pattern” and “Miniopterus pattern”, in consideration of sperm differentiation process and change in adnexal organs (Gustafson 1979). In particular, studies of serial events in testicles and sperm fate and loss in epididymis during the male reproductive cycle, conducted on temperate hibernating bats, are worthy of notice (Lee 2018a, 2018b).

In terms of reproduction in female bats, mating, ovulation, fertilization, egg development and delivery occur unremitted in the tropical non-hibernating bats as same to the general mammalian types. The tropical non-hibernating bat species include Desmodus rotundus mutinus (Wimsatt & Trapido 1952), Pteropus giganteus, Cynopterus sphinx, C. gangeticus, Rhinopoma kinneari, Megaderma lyra, Rhinolophus rouxi, Hipposideros bicolor pallidus, H. speoris, Taphozous longimanus and Scotophilus wroughtoni (Gopalakrishna & Moghe 1960; Gopalakrishna et al. 1974).

As a strategy to adapt to environmental changes, the temperate female bats have evolved by unique reproduction of their own, including “prolonged sperm storage type” and “delayed implantation type”.

First, the prolonged sperm storage type refers to that the sperms stored in the female reproductive tract have long-term hibernation after mating in autumn, followed by ovulation, fertilization and embryonic development in consecutive order in spring of next year, and includes vespertilionine from North America (Guthrie 1933; Reeder 1939; Christian 1956; Krutzsch 1975), and temperate vespertilionids and rhinolophids (Matthews 1937; Wimsatt 1942; Hiraiwa & Uchida 1955, 1956; Wimsatt et al. 1966; Racey & Potts 1970; Gopalakrishna & Madhavan 1971; Medway 1972; Mōri & Uchida 1974; Krishna & Dominic 1978; Racey 1979; Uchida et al. 1984, 1988; Son et al. 1987, 1988; Mōri et al. 1989; Lee & Son 2000; Sharifi et al. 2004).

Second, the delayed implantation type refers to that ovulation and fertilization occur after mating in autumn and the fertilized egg is stored in the female reproductive tract while embryogenesis occurs consecutively in the spring of the next year. This type includes Miniopterus schreibersii and Eidolon helvum (Mutere 1965) and Rhinolophus rouxi (Ramakrishna & Rao 1977) of vespertilionid, as well as Miniopterus schreibersii fuliginosus (Uchida & Mōri 1974a; Mōri & Uchida 1980, 1981a, 1981b, 1982; Kimura & Uchida 1983, 1984).

Fragmentary studies of reproductive phenomenon in female bats were reported (Uchida 1950, 1953; Hiraiwa & Uchida 1955, 1956; Wimsatt & Parks 1966; Wimsatt et al. 1966; Racey & Potts 1970; Mōri & Uchida 1974, 1980, 1981a, 1981b, 1982; Uchida & Mōri 1974a, 1974b; Racey 1979; Oh et al. 1985; Son et al. 1987, 1988; Uchida et al. 1988; Mōri et al. 1989; Kim & Oh 1991; Lee & Son 2000), while an annual reproductive cycle in the female Rhinolophus ferrumequinum korai was not conducted. Accordingly, this study was conducted to examine serial reproductive cycles including morphological changes and characteristics in female reproductive tract as well as sperm storage and loss in reproductive tract per month and site.

Materials and methods

Experimental animals were collected and examined under the guidelines of the Kyungnam University Institutional Animal Care and Use Committee (KUIAC).

In this study, 58 female Rhinolophus ferrumequinum korai entities collected at the same date every month as possible January 2017-December 2018 in the abandoned mines over Gyeongnam and Jeonnam regions were used as study materials (Table I), transferred to the laboratory immediately after collection and inhalation-anesthetized with ether before use. The tissues collected for monthly observation of morphological characteristics and changes in reproductive tract were washed immediately with Milloning’s buffer (4°C, pH 7.4) solution. After completion of washing, the tissues were soaked in a 3%-Glutaraldehyde solution (4°C, pH 7.4, Milloning’s buffer) for 24 h and photographed (Figure 1). For observation with scanning electron microscope, the reproductive tract of each month was transected with the razor and fixed in 3% Glutaraldehyde solution for 24 h. Post-fixation tissues were washed with Milloning’s buffer solution twice in 20-min intervals and fixed in 1.33% OsO₄ solution after 2 h. Post-fixation tissues were washed with Milloning’s buffer solution twice in 20-min intervals. Post-washing tissues were dehydrated twice in 30-min intervals in alcohol concentration-ascending order (70%, 80%, 85%, 90%, 95% and 100%, respectively). Post-dehydration tissues were transposed into HMDS (hexamethyl-disilazane, Sigma) solution twice for 24 h, respectively. Post-transposition tissues were evaporated to OsO₄ steam within the Osmium coater (HPC-1 S, Japan) at 10–13 PS (10 x 0.1 Torr) vacuum and 3 mA for 15 s. Post-evaporation tissues were observed by the scanning electron microscope (TM-1000, Hitachi, Japan). Hematoxylin & Eosin staining and Trichrome staining were conducted by using the conventional paraffin sectioning method in each tissue, respectively, to understand the histological characteristics in the reproductive tract per year. For the transmission electron microscopic observation, tissues by the site of the female reproductive tract were sectioned in 1.5–2 mm³ size, pre-fixed in 3% Glutaraldehyde solution for 24 h, washed with the same buffer (Milloning’s buffer) twice in 20-min intervals, and immediately post-fixed in 1.33% OsO₄ solution for 2 h. Post-fixation tissues were washed with the same buffer twice in 20-min intervals. Post-washing tissues were dehydrated twice in 30-min intervals in alcohol concentration-ascending order (70%, 80%, 85%, 90%, 95% and 100%, respectively). The tissues dehydrated were transposed to propylene oxide solution and embedded with Epon 812 synthetic resins. The tissues embedded were cut in 400 nm thickness by ultramicrotome (MT-1; Sorvall, Dupont), stained with 0.5% toluidine blue and observed, from which consecutive fragments were obtained in 60–70 nm thickness to be double-stained with Uranyl acetated and lead citrate solutions and observed by transmission electron microscope (TEM, H-600, Hitachi).

Table I. Sperm storage and disappearance per month and the site in the female reproductive tract in Korean greater horseshoe bats (Rhinolophus ferrumequinum korai)

		Uterus (U)				Uterotubal junction (Utj)
Date examined	Number of bats	Uterine lumen (Ul)		Uterine gland (Ug)		Colliculus tubaricus (Ct)		Intramural part (Ip)		Caudal isthmus (Cui)
Aug. 15, 2017	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Aug. 25, 2018	(2)	S: +	L: †	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Sep. 12, 2017	(2)	S: + +	L: † †	S: +	L: †	S: +	L: o	S: +	L: o	S: +	L: o
Sep. 23, 2018	(3)	S: + +	L: † †	S: +	L: †	S: +	L: o	S: +	L: o	S: +	L: o
Oct. 22, 2018	(2)	S: + +	L: † †	S: + +	L: †	S: +	L: o	S: +	L: o	S: +	L: o
Oct. 25, 2018	(3)	S: + +	L: † †	S: + +	L: † †	S: +	L: o	S: +	L: o	S: +	L: o
Nov. 22, 2017	(2)	S: + + +	L: † † †	S: + +	L: † †	S: +	L: o	S: +	L: o	S: + +	L: o
Nov. 23, 2018	(3)	S: + + +	L: † † †	S: + +	L: † †	S: +	L: o	S: +	L: o	S: + +	L: o
Dec. 18, 2017	(3)	S: + + +	L: † † †	S: + +	L: † †	S: +	L: o	S: +	L: o	S: + + +	L: o
Dec. 23, 2018	(3)	S: + + +	L: † † †	S: + +	L: † †	S: +	L: o	S: +	L: o	S: + + +	L: o
Jan. 18, 2017	(2)	S: + +	L: † †	S: +	L: †	S: +	L: o	S: +	L: o	S: + + +	L: o
Jan. 23, 2018	(2)	S: + +	L: † †	S: +	L: †	S: +	L: o	S: +	L: o	S: + + +	L: o
Fab. 18, 2017	(3)	S: + (a few)	L: † (a few)	S: + (a few)	L: † (a few)	S: +	L: o	S: +	L: o	S: + + +	L: o
Fab. 25, 2018	(3)	S: + (a few)	L: † (a few)	S: -	L: † (a few)	S: +	L: o	S: +	L: o	S: + + +	L: o
Mar. 8, 2017	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Mar. 25, 2018	(3)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Apr. 18, 2017	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Apr. 25, 2018	(3)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
May 11, 2017	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
May 28, 2018	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Jun. 8, 2017	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Jun. 18, 2018	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Jul. 14, 2017	(2)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o
Jul. 24, 2018	(3)	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o	S: -	L: o

The symbols of -, +, ++, and +++ in the sites of sperm storage show the absence of sperm, presence of small, moderate and large numbers of sperm, respectively. The symbols of ○, †, † †, and † † † in the sites of reproductive tract show absence of leucocytes, presence of small, moderate, and large numbers of leucocytes, respectively. L, leucocytes; S, sperm.

Figure 1. Morphological change in the female reproductive tract in Korean greater horseshoe bats (Rhinolophus ferrumequinum korai). The size of the uterus was maintained consistently from the end of August to the end of March of the next year, increased gradually since the end of March when ovulation and fertilization occurred and reached a maximum in May. Meanwhile, it decreased remarkably during the delivery and nurturing period. F, fetus; O, ovary; Ov, oviduct; U, uterus; V, vagina; (a), late-August; (b), late-September; (c), late-October; (d), late-November; (e), late-December; (f), late-January; (g), late-February; (h), late-March; (i), late-April; (j), late-May; (k), early-June; (l), late-July

Results

To understand female the reproductive cycle of the Korean hibernating horseshoe bats (R. ferrumequinum korai), morphological changes in the reproductive tract per month and prolonged sperm storage and sperm loss per site (uterine lumen, uterine gland, vagina, uterotubal junction and caudal isthmus of the oviduct) were investigated and the results are below (Tables I and II; Figures 1–7).

Table II. Comparison of characteristics of female annual reproductive cycle by step and sperm survival, storage, and disappearance by site in 14 species of Vespertilionidae and 2 species of Rhinolophidae

Species	Period					Region of Sperm Storage			Authors
	Copulation	Ovulation & fertilization	Pregnancy	Parturition	Nursing	U	Utj	Cui
Miniopterus australis	mid-October (in northern hemisphere)	Ovulation and fertilization occur after mating.	__	__	__	*	*	*	(11)
M. schreibersii	late May/early June (in south hemisphere)	Ovulation and fertilization occur after mating.	__	__	__	*	*	*	(9), (11), (13)
M. schreibersii fulignosus	Autumn	Ovulation takes place a few days following fall copulation.	__	__	__	death	+, alive	+, a few	(14), (15), (16), (17)
Murina leucogaster	Autumn	*	__	__	__	alive	+, alive	no sperm	(21)
Myotis formosus tsuensis	late October	Ovulation occur after arousal from hibernation in spring.	__	__	__	+, alive (a few)	+, alive (many sperms)	*	(19)
M. ricketti	Copulation occurred from Oct. to Apr.	Ovulation was delayed until arousal from hibernation (in May and June).	__	__	__	*	*	*	(23)
Pipistrellus abramus	late October	*	6 months (in uterus)	*	*	+, alive	__	__	(8), (10), (20)
P. abramus abramus	late October	late April	118 days (in uterus)	*	*	+, alive or death	__	__	(3), (4)
P. ceylonicus chrysoyhrix	*	*	*	*	*	+, alive	__	+, alive	(6)
P. pipistrellus	*	*	*	*	*	+, alive	__	__	(5)
P. tralatitius abramus	Autumn	late April	pre/or after about 70 days	early July	__	+, alive	*	*	(1)
	late October	late April	pre/or after about 70 days	early July	__	+, alive	__	__	(2)
Scotophilus heathi	*	*	*	*	*	+, alive	__	+, alive	(12)
Tylonycteris pachypus	*	*	*	*	*	*	__	+, alive	(7)
T. robustula	*	*	*	*	*	+, alive	__	+, alive	(7)
Rhinolophus ferrumequinum nippon	mid or late October	early April	*	*	*	death	Ip (a few): +, alive	+, alive	(18)
R. ferrumequinum korai	early October	late April	*	*	*	death	Ip (a few): +, alive	+, alive	(22)
R. ferrumequinum korai	late August to early September	Ovulation and fertilization occurs from late March.	late March to late May	early to mid-June	early June to mid- August	death	Ip (a few): +, alive	+, alive	(24)

(1), Uchida (1950); (2), Uchida (1953); (3), Hiraiwa and Uchida (1955); (4), Hiraiwa and Uchida (1956); (5), Racey and Potts (1970); (6), Gopalakrishna and Madhavan (1971); (7), Medway (1972); (8), Mōri and Uchida (1974); (9), Uchida and Mōri (1974a); (10), Uchida and Mōri (1974b); (11), Richardson (1977); (12), Krishna and Dominic (1978); (13), Wallace (1978); (14), Mōri and Uchida (1980); (15), Mōri and Uchida (1981a); (16), Mōri and Uchida (1981b); (17), Mōri and Uchida (1982); (18), Oh et al. (1985); (19), Son et al. (1987); (20), Uchida et al. (1988); (21), Mōri et al. (1989); (22), Lee and Son (2000); (23), Wang et al. (2008); (24), This paper; U, uterus; Utj, uterotubal junction; Cui, caudal isthmus of the oviduct; +, stored sperm; -, no sperm; *, no data; Ip, intramural part of the uterotubal junction.

Figure 2. Optical, scanning electron microscopic and transmission electron microscopic images of virginal lumen, cervix, uterus and uterine lumen (August sample). Attention should be focused on the figure of sperm mixed by secretion from vaginal epithelium in the vaginal lumen (Figure 2(a)) and phagocytosed by leukocytes in the uterine lumen (Figure 2(b, c)). L, leucocyte; S, sperm; Sh, sperm head; Uc, uterine cervix; Ucl, uterine cervix lumen; Ug, uterine gland; Ul, uterine lumen; Uw, uterine wall; V, vagina. (a), SEM; (b), Hematoxylin & Eosin stain; (c), TEM

Figure 3. Optical microscopic, scanning electron microscopic and transmission electron microscopic images of sperm killing in the uterine lumen and uterine gland (December sample). Sperm were phagocytosed by leukocytes in the uterine lumen (Figure 3(a-c)) and uterine gland (Figure 3(d)). Ec, endometrial epithelial cell; L, leucocyte; S, sperm; Sh, sperm head; Ug, uterine gland; Ul, uterine lumen; Uw, uterine wall; (a), Hematoxylin & Eosin stain; (b), SEM; (c), TEM; (d), toluidine blue stain

Figure 4. Scanning electron microscopic and transmission electron microscopic images of sperm kill in uterine lumen and uterine gland. In the experimental group around the last 10 days of January, a large number of sperm was phagocytosed by leukocytes in the uterine lumen (Figure 4(a, b)) and uterine gland (Figure 4(c)). In the experimental group around the end of February, an extremely small number of sperm were phagocytosed by leukocytes in the uterine lumen (Figure 4(d, e)). In particular, in the experimental group in February, no sperm was observed in the uterine gland as a result of phagocytosis by leukocytes (Figure 4(f)). Ec, endometrial epithelial cell; L, leucocyte; Ly, lysosome; Mv, microvilli; N, nucleus; S, sperm; Sh, sperm head; Ug, uterine gland; Uge, uterine gland epithelium; Ul, uterine lumen. (a, c, d and f), SEM; (b, e), TEM

Figure 5. Images of the uterus before ovulation and ovulated ovum, and post-delivery litters and the uterus during the nurturing period. Endometrium was wrinkled approximately at the end of March before ovulation (Figure 5(a)). Note the ovulated ovum (Figure 5(b)) and the appearance of the cub after giving birth (Figure 5(c)). While the uterus approximately the end of July was not wrinkled but even (Figure 5(d)). In particular, it should be noted that no sperm were observed in the uterine lumen before ovulation (the early of March) (Figure 5(a)) and during the nurturing period (the end of July) (Figure 5(d)). Cc, cumulus cell; N, nucleus; Ul, uterine lumen; z, zona pellucida. (a), early-March; (b), late-March; (c), mid-June; (d), late-July

Figure 6. Optical and scanning electron microscopic images of the caudal isthmus of oviduct and sperms stored Therein. It should be noted that no sperm was observed in the caudal isthmus of the oviduct (Figure 6(a, b)). The epithelium of the caudal isthmus of the oviduct consists of ciliated epithelial cells and non-ciliated epithelial cells (Figure 6(c)). It should be noted that sperm were stored between the ciliated epithelial cells of the caudal isthmus of the oviduct (Figure 6(d)). Ce, ciliated epithelial cell; Cil, caudal isthmus lemen; Ne, non-ciliated epithelial cell; Oe, oviductal epithelium; S, sperm; (a), hematoxylin & Eosin stain; (b-d), SEM; (a and b), mid-August; (c), mid-September; (d), late-December

Figure 7. General mimetic diagram of relationship between sperm storage and loss in reproductive tract during female annual reproductive cycle of Korean hibernating greater horseshoe bats (R. ferrumequinum korai). The reproductive cycle is divided into six stages. The first stage is the mating period (the end of August). The second stage is the cleansing period (from the end of August to the end of February of the next year). During the cleansing period, some of sperm ejaculated after mating are phagocytosed and killed by leukocytes and this process proceeds gradually until February of the next year. From ovulation to the gestation and nurturing period, no sperm exist in the uterine lumen and uterine gland (Table I, Figure 5(a, d)). Sperm survival and storage occur in the caudal isthmus of the oviduct. The third stage is from the end of August to the end of September. When sperm in the uterine lumen move towards the uterotubal junction and are stored in the caudal isthmus of the oviduct. Only the sperm reaching the caudal isthmus of oviduct were found to be engaged in fertilization in the next year, and the duration of sperm storage was approximately 170 days. The fourth stage is the ovulation and fertilization period (the end of March). The fifth stage is the gestation period (from the end of March to the end of May) (approximately 70 days). The sixth stage is delivery (early and mid-June) and the nurturing period (from the end of June to mid-August) (nurturing period: approximately 80 days). †When sperm in uterine lumen move towards the uterotubal junction

Morphological changes and characteristics of female reproductive tract per month

Change in the size of the uterus

There was no change in size of the uterus observed from the mating period (the end of August) to ovulation and implantation period (approximately the end of March) (Figure 1(a-h)). Meanwhile, the size of the uterus expanded during the gestation period, April (from the end of March to the end of May) (Figure 1(i)), reaching a maximum in May (Figure 1(j)). The size of uterus was reduced remarkably during the delivery period (early and middle of June) and the nurturing period in June (Figure 1(k)) and July (Figure 1(l)).

Prolonged sperm storage and sperm fate in female reproductive tract per month

Sperm fate in the uterine lumen, uterine gland and vagina

Approximately at the end of August, the mating period, some of the sperm ejaculated immediately after mating were phagocytosed by secretion of vaginal epithelial cells (Figure 2(a)). In particular, an extremely small number of sperm was phagocytosed by leucocytes (Figure 2(b, c)), while penetration and satiating activity by leucocytes were not observed from the uterine gland (Table I). A greater number of sperm were phagocytosed by leucocytes in the uterine lumen approximately at the end of September, compared to the experimental group in August, while an extremely small number of sperm was phagocytosed by leucocytes in the uterine gland (Table I). Phagocytosis by leucocytes was observed from the uterine lumen and uterine gland approximately at the end of October, compared to that at the end of September (Table I). In November and December, phagocytosis by leucocytes reached a peak in the uterine lumen and uterine gland (Table I; Figure 3(a–d)). During January of the next year, a number of phagocytosis by leucocytes was observed from the uterine lumen and uterine gland (Table I; Figure 4(a–c)). Meanwhile, during February, phagocytosis by leucocytes caused an extremely small number of sperm to be killed by leucocytes in the uterine lumen (Table I; Figure 4(d, e)). Differing from the experimental group of January, only the leucocytes existed in the uterine gland (Table I, Figure 4(f)). Such phagocytosis by leucocytes in the uterine lumen was observed from the end of August to the end of February in the next year (Table I). Sperm were not observed from the uterine lumen until ovulation (early and middle of March) (Figure 5(a); Table I). Ovulation occurred approximately at the end of March (Figure 5(b); Table II), and delivery occurred approximately at the early and middle of June (Figure 5(c); Table II). During the nurturing period (from the early of June to middle August), sperm were not observed from the uterine lumen (Figure 5(d); Table I).

Sperm fate in the uterotubal junction

The uterotubal junction, the entry to the oviduct from the uterus, divided into two parts, colliculus tubaricus and intramural part of the uterotubal junction was observed, and the result thereof is below.

Situation in the colliculus tubaricus

The colliculus tubaricus is the first gate where sperm ejaculated after mating pass the uterus and moving to the oviduct, referring to the well-developed site in which the oviduct is stretched to the uterine lumen. According to the result of this study, sperm failed to reach the colliculus tubaricus approximately the last 10 days of August, the starting point of mating (Table I). A number of sperm reached the colliculus tubaricus September–February of the next year and no phagocytosis by leucocytes was observed (Table I). Also, sperm were not observed from the colliculus tubaricus approximately at the end of March when ovulation and implantation occurred, as well as penetration by leucocytes and sperm were not observed from the colliculus tubaricus during the gestation (from the end of March to the end of May) and nurturing (June and July) periods and until August (Table I).

Situation in the intramural part of the uterotubal junction

The situation in the intramural part of the uterotubal junction led to almost same result as the situation in the colliculus tubaricus described above (Table I). The only difference was that an extremely small number of sperm was observed from the colliculus tubaricus and intramural part of the uterotubal junction during January and February, while no sperm was observed from the intramural part of the uterotubal junction from March to the end of August (Table I).

Sperm storage in the caudal isthmus of the oviduct

Some of the sperm that ejaculated during the mating-starting period (the end of August) was captured by the secretion of vaginal epithelial cells (Figure 2(a)), among which some flowing into the uterus were killed by leukocytes (Figure 2(b, c)). Most of sperm ejaculated into the uterus approximately in August failed to reach the caudal isthmus of the oviduct (Table I; Figure 6(a, b)). In the experimental group from March to August, sperm were not observed from the caudal isthmus of the oviduct (Table I).

However, sperm alive in the uterus (in the experimental group September-mid-February) passed through the uterotubal junction and reached the caudal isthmus of the oviduct in which it was observed as alive for long-term hibernation (Table I; Figure 6(d)), with no presence of leukocytes (Table I).

Discussion

Morphological changes and characteristics of female reproductive tract per month

According to the results of this study, there was no major change in the size of the uterus from the end of August (mating-starting period) (Figure 1(a)) to March of the next year (Figure 1(h)). Meanwhile, remarkable change appeared approximately at the end of April (Figure 1(i)). In other words, the appearance of the uterus largely expanded in April, resulting from fertilization by sperm and egg ovulating approximately the end of March already and consecutive embryogenesis. The pregnancy period was approximately 70 days (from the end of March to the end of May (Figure 1(h-j)), and delivery occurred approximately early and mid-June (Figures 1(k), 5(c) and 7; Table II). Approximately the early of June, immediately after delivery, the size of the uterus remarkably reduced (Figure 1(k)). It suggests that this period is for nurturing (approximately 80 days) when much energy is consumed to nurture litters.

Sperm storage and fate in the female reproductive tract

As shown in Table II, during hibernation, sperm survived in the uterus for a number of months and fertilized with the egg, ovulated in spring of the next year while attaining fertility capacity in Pipistrellus tralatitius abramus (Uchida 1950, 1953), P. abramus abramus (Hiraiwa & Uchida 1955, 1956), P. abramus (Mōri & Uchida 1974; Uchida & Mōri 1974b; Uchida et al. 1988), P. pipistrellus (Racey & Potts 1970) belonging to Vespertilionidae. This phenomenon also appears in Myotis lucifugus, Epitesicus fuscus (Wimsatt 1942, 1944). Accordingly, Uchida and Mōri (1974b) have reported that infiltration by leukocytes does not occur in the uterus where sperms are stored during the winter and that the intrauterine accumulation of sperm plays a crucial role in the survival of an appropriate number of sperm by limiting contact between sperm and leukocytes penetrating into the uterine lumen and as the site where acrosome reaction is induced. Mōri and Uchida (1974) describe that sperm are digested as a part of the phagocytosis by epithelial cells of fallopian tube in Japanese house bats, Pipistrellus abramus.

Meanwhile, survival and storage of sperm in the uterus and caudal isthmus of the oviduct have been reported in Indian P. ceylonicus chrysothrix (Gopalakrishna & Madhavan 1971) and Tylonycteris pachypus, T. Robustula (Medway 1972) as well as Scotophilus heathi (Krishna & Dominic 1978). Also, almost no leukocyte were reported in P. pipistrellus, Nyctalus noctula, Myotis nattereri and M. Daubentoni as well as Rhinolophus ferrumequinum, R. Hipposideros (Racey 1975, 1979). In South African Pipistrellus rusticus species, no leukocyte was found in the oviduct and uterine lumen and some of the sperm may be phagocytized by uterine epithelial cells while a number of sperm may be rapidly lost, suggesting the possibility of exposure from the vagina after fertilization (Van der Merwe & Rautenbach 1990). According to Mattner (1968, 1969a), the uterus where leucocytes penetrate and sperm storage are spatially separated from each other per animal species, which has effects in that sperm are protected from phagocytosis by leucocytes and a sufficient number of sperm necessary for fertilization is secured. Meanwhile, the caudal isthmus of the oviduct is the only space for sperm storage in Myotis formosus tsuensis species (Son et al. 1987), suggesting that the caudal isthmus of the oviduct is closely related to sperm survival when considering that sperm exist between non-ciliated epithelial cells in the caudal isthmus of the oviduct (Figure 6(c)).

Meanwhile, sperm storage occurs in the UTJ (uterotubal junction) and the caudal isthmus of the oviduct, respectively, in P. savii velox and P. endoi (Son et al. 1988), in both of which sperm death are caused in the uterus. As influential factors on prolonged sperm survival and sperm storage, fructose detected from endometrial cells and a great volume of glycogen from non-ciliated epithelial cells in the caudal isthmus of the oviduct (Pipeistrellus: Racey 1975; Myotis lucifugus, M. velifer: Crichton et al. 1981; Rhinolophus ferrumequinum korai: Lee & Son 2000) serve as a nutrient source to prolong sperm storage as well as influence the ovary and prolonged survival of Graafian follicles (Wimsatt & Kallen 1957; Wimsatt & Parks 1966; Son et al. 1987). Additionally, Son et al. (1988) imply that characteristics and amount of secretory granules in epithelial cells are closely related to the amount of sugar molecules covering the epithelial microvilli. For the Murina leucogaster species, the major site of sperm storage is the uterotubal junction; the sperm that head towards the non-ciliated epithelial cells are closely related to the well-developed microfibril the amount of sugar molecules after ovulation are swallowed by non-ciliated epithelial cells covered by a large amount of glycocalyx (Mōri et al. 1989).

Meanwhile, for R. ferrumequinum nippon (Mōri et al. 1982; Oh et al. 1985) R. ferrumequinum korai (Kim & Oh 1991; Lee & Son 2000) which belong to Rhinolophus, sperm are killed in the uterus and an extremely small number of sperm survive and are stored in the intramural part of the uterotubal junction (R. ferrumequinum insulanus: Racey 1975, 1979; R. ferrumequinum korai: Lee & Son 2000). In this study, an extremely small number of sperm were observed in the colliculus tubaricus and intramural part of the uterotubal junction (Table I). However, most of the sperm were reported surviving in the caudal isthmus of the oviduct (Gopalakrishna & Madhavan 1971; Medway 1972; Krishna & Dominic 1978; Mōri & Uchida 1980, 1981a, 1981b, 1982; Oh et al. 1985; Lee & Son 2000). A similar result was obtained in this study (Table I).

Meanwhile, for Miniopterus schreibersii fuliginosis of delayed implantation type, leukocytes were noted to penetrate the uterus before mating (Uchida & Mōri 1974a); however, it was found to penetrate the uterus immediately after mating in this study (Table I; Figure 2 (inset b, c)). For M. schreibersii fuliginosis, intrauterine sperms were all killed and the uterotubal junction and caudal isthmus of the oviduct were determined as the space for sperm survival and storage (Uchida & Mōri 1974a; Mōri & Uchida 1980, 1981a, 1981b, 1982). Additionally, ovulation and fertilization occurred for the first time a number of days after mating (Mōri & Uchida 1980, 1981a, 1981b, 1982). A similar result was obtained from this study (Figures 5(b) and 7).

For the Pipistrellus abramus (Uchida & Mōri 1974b), Myotis lucifugus, and M. velifer (Krutzsch et al. 1982), phagocytosis by sperm is caused by endometrial epithelial cells and phagocytes during the hibernation period. Based on this fact, Krutzsch et al. (1982) highlight that sperm survival should be based on the capacity of sperm in the female reproductive system. However, according to this study, sperm were gradually killed by leukocytes in the uterine lumen, secretion in the uterine gland, and phagocytosis by leukocytes (Figures 2(b, c), 3(a-d) and 4(a-f)). Such phagocytosis is to keep the intrauterine environment clean and facilitate implantation of the fertilized egg. This period may be considered a cleansing period (Figure 7).

There are three barriers against sperm transfer, including the uterine cervix, uterotubal junction and caudal isthmus of the oviduct in the female reproductive system (Hafez 1974), in which unnecessary matters as well as the destroyed sperms were removed from the fertilization site and female reproductive system. A number of study results showed that sperm were removed by enzyme lysis and phagocytosis caused by polymorphonuclear neutrophilic leucocytes in the vagina (Beaver 1960; Howe & Black 1963; Phillips & Mahler 1977a, 1977b) and uterus (Austin 1957, 1960), phagocytosis by leukocytes penetrated the uterine cervix (Mattner 1969a, 1969b), oviduct (Howe 1967), etc., and absorptive action by the oviductal epithetial cells (Zamboni 1971) and cervical epithelium (Rasweiler 1987). Meanwhile, for Japanese greater horseshoe bats (R. ferrumequinum nippon), sperm are phagocytized and digested by fibroblasts. Thereafter, sperm are deposited in the uterine lumen in addition to a large amount of secretion from the endometrial connective tissues infiltrated by polymorphonuclear neutrophilic leucocytes (Uchida et al. 1984). Sperm not involved in fertilization were removed by phagocytosis by macrophage (Oxberry 1979; Koyanagi & Nishiyama 1981). As mentioned earlier, there is a difference in sperm killing sites by species, considered to contribute to reproductive cycling by species.

Thus, the breeding cycle of a species should be explained on the basis of the breeding phenomenon in females and males. In other words, for the reproductive cycle of hibernating Korean greater horseshoe bats (R. ferrumequinum korai), mating is initiated August–September when spermatogenesis is active in males (Lee 2018a). During long-term hibernation after mating, immature sperm cells left in the male seminiferous tubule are completely removed by phagocytosis by Sertoli cells, which is called the cleansing period. Such phagocytosis suggests the preparation period to produce new sperm from testicles (Lee 2018a). Additionally, sperm left in the epididymis after mating and ejaculation are also removed by phagocytosis by leukocytes as well as secretion from testicular epithelium, also called the cleansing period to prepare to accept new sperm the next year by removing extra sperm left in the epididymis (April–June) when the first removal process occurs (Lee 2018b). As the second sperm removal process (July–August), purification occurs to be ready for maintaining mature sperm only by removing the deformed sperms introduced from the testicles and other residues (Lee 2018b). It is considered as a breeding strategy to improve fertility.

In the serial process of change occurring in the female reproductive tract, some of the sperm ejaculated to the uterus during the mating period (the end of August) are removed by phagocytosis of leucocytes. Such a process occurs until the end of February of the next year and is considered as to maintain the cleanliness of the intrauterine environment to facilitate implantation of the fertilized egg. Accordingly, this period may be called the cleansing period (approximately 190 days). Consecutive embryogenesis (Figure 7) proceeds, as survived sperm among them are stored in the caudal isthmus of the oviduct during long-term hibernation, moved to the fertilization site the next year, and infuse with the egg for fertilization (Figure 7; Table II). At this point, sperm storage, survival, and transfer in the female reproductive tract have a major impact on fertilization (Son et al. 1987). Also, according to this study, sperm initially infiltrated to the inside of the female reproductive tract are recognized as foreign materials and killed by leukocytes to maintain an appropriate number of sperm, considered as a physiological mechanism to prevent polyspermy and improve fertility. Fertilization may be favorable for the mid or late sperm group compared to the initial sperm group ejaculated inside of the female reproductive tract to infuse with the egg.

Accordingly, the female annual reproductive cycle in the Korean greater horseshoe bat (R. ferrumequinum korai) was classified as: (1) Mating period (the end of August), (2) Cleansing period (from the end of August to the end of February of the next year), (3) Sperm transfer period (when the intrauterine sperm move towards the uterotubal junction to be stored in the caudal isthmus of the oviduct), (4) Ovulation and fertilization period (the end of March), (5) Pregnancy period (from the end of March to the end of May) and (6) delivery (early and mid-June) and nurturing period (June-mid August). In particular, only the sperm reaching the caudal isthmus of the oviduct as the space for sperm storage engages in fertilization. For the annual female reproductive cycle in R. ferrumequinum korai, mating and ovulation as well as fertilization, implantation, delivery and the nurturing period occur approximately 1 month earlier than Rhinolophidae bats including most of the species belonging to Vespertilionidae (Table II), suggesting that such change and difference in reproductive cycle may contribute to photoperiod and temperature change as insisted by Lee (2018a, 2018b).

In conclusion, change in the size of the uterus during the female reproductive cycle is strongly related to the mating and delivery periods in hibernating Korean greater horseshoe bats (R. ferrumequinum korai). In particular, initial sperm that had been ejaculated into the female reproductive tract since the end of August were phagocytosed and killed by phagocytosis and leukocytes that had infiltrated into the uterus until the end of February of the next year. Such phagocytosis seemed to result in maintaining a clean intrauterine environment to facilitate of implantation of the fertilized egg. Also, phagocytosis by leukocytes was considered a physiological mechanism to kill the sperm penetrating the female reproductive tract by recognizing them as alien materials, to maintain the appropriate number of sperm at the fertilization site of the fallopian tubes, thus improving fertility. Accordingly, this period may be called the cleansing period (approximately 190 days from the end of August to the end of February of the next year). The sperm became extinct mainly in the uterine lumen, and sperm became extinct by secretion from the uterine gland and the phagocytosis by leukocytes.

Only the sperm reaching the caudal isthmus of the oviduct were found to be involved in fertilization in the next year. The sperm were stored at the caudal isthmus of oviduct for approximately 170 days. Ovulation and fertilization occurred at the end of March while embryogenesis appeared consecutively with implantation. The pregnancy period was approximately 70 days; delivery occurred around early and mid-June; and the nurturing period was approximately 80 days.

Geolocation information

Country: Korea, Republic of Region: Gyeongsangnam-do City: Changwon-si

Latitude: N35°11′ (Masan-si)

Longitude: E128°34′ (Masan-si)

Disclosure statement

No potential conflict of interest was reported by the author.

Additional information

Funding

This work was supported by a 2019 Kyungnam University Foundation Grant [20190099].